Serum albumin-binding fibronectin type iii domains

ABSTRACT

The present invention relates to polypeptides which include tenth fibronectin type III domains ( 10 Fn3) that binds to serum albumin, with south pole loop substitutions. The invention further relates to fusion molecules comprising a serum albumin-binding  10 Fn3 joined to a heterologous protein for use in diagnostic and therapeutic applications.

REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/968,181 entitled “Novel Serum Albumin-Binding Fibronectin Type IIIDomains” filed on Mar. 20, 2014, the contents of which are herebyincorporated by reference in its entirety.

BACKGROUND

Inadequate half-lives of therapeutics often necessitate theiradministration at high frequencies and/or higher doses, or the use ofsustained release formulations, in order to maintain serum levelsnecessary for therapeutic effects. Yet, this is often associated withnegative side effects. For example, frequent systemic injections presentconsiderable discomfort to the subject, and pose a high risk ofadministration-related infections, and may require hospitalization orfrequent visits to the hospital, in particular when the therapeutic isto be administered intravenously. Moreover, in long term treatments,daily intravenous injections can also lead to considerable side effectsof tissue scarring and vascular pathologies caused by the repeatedpuncturing of vessels. Similar problems are known for all frequentsystemic administrations of therapeutics, such as, for example, theadministration of insulin to diabetics, or interferon drugs to patientssuffering from multiple sclerosis. All these factors lead to a decreasein patient compliance and increased costs for the health system.

This application provides compounds that increase the serum half-life ofvarious therapeutics, compounds having increased serum half-life, andmethods for increasing the serum half-life of therapeutics. Suchcompounds and methods for increasing the serum half-life of therapeuticscan be manufactured in a cost effective manner, possess desirablebiophysical properties (e.g., Tm, substantially monomeric, orwell-folded), and are of a size small enough to permit tissuepenetration.

SUMMARY

The invention is based, at least in part, on the discovery of novelsouth pole-based serum albumin binding fibronectin type III tenth domain(¹⁰Fn3) containing Adnectins (PKE2 Adnectins), which provide enhancedproperties over prior north pole-based serum albumin binding ¹⁰Fn3domain containing Adnectins.

In one aspect, the invention provides a polypeptide comprising a ¹⁰Fn3domain, wherein the ¹⁰Fn3 domain comprises a) AB, BC, CD, DE, EF, and FGloops, b) a CD loop with an altered amino acid sequence relative to thesequence of the corresponding CD loop of the human ¹⁰Fn3 domain, and c)wherein the polypeptide binds to human serum albumin with a K_(D) ofless than 500 nM.

In certain embodiments, the ¹⁰Fn3 domain further binds to one or more ofrhesus serum albumin, cynomolgus serum albumin, mouse serum albumin, andrat serum albumin. For example, the ¹⁰Fn3 domain may bind to HSA, rhesusserum albumin, and cynomolgus serum albumin, or the ¹⁰Fn3 domain maybind to HSA, rhesus serum albumin, cynomolgus serum albumin, mouse serumalbumin, and rat serum albumin. In some embodiments, the ¹⁰Fn3 domainbinds to the corresponding serum albumin with a K_(D) of less than 500nM, for example, a K_(D) of less than 100 nM, or even a K_(D) less than10 nM. In some embodiments, the ¹⁰Fn3 domain binds to serum albumin at apH range of 5.5 to 7.4.

In certain embodiments, the ¹⁰Fn3 domain binds to domain I-II of HSA.

In certain embodiments, the serum half-life of the polypeptidecomprising the ¹⁰Fn3 domain in the presence of human serum albumin is atleast 10 hours, such as at least 20 hours, or at least 30 hours.

In certain embodiments, the CD loop comprises an amino acid sequenceaccording to the formulaG-X₁-X₂-V-X₃-X₄-X₅-S-X₆-X₇-G-X₈-X₉-Y-X₁₀-X₁₁-X₁₂-E (SEQ ID NO: 170),wherein,

-   -   (a) X₁ is selected from the group consisting of R or W;    -   (b) X₂ is selected from the group consisting of H, E, D, Y, or        Q;    -   (c) X₃ is selected from the group consisting of Q or H;    -   (d) X₄ is selected from the group consisting of I, K, M, Q, L,        or V;    -   (e) X₅ is selected from the group consisting of Y, F, or N;    -   (f) X₆ is selected from the group consisting of D, V, or E;    -   (g) X₇ is selected from the group consisting of L, W, or F;    -   (h) X₈ is selected from the group consisting of P or T;    -   (i) X₉ is selected from the group consisting of L or M;    -   (j) X₁₀ is selected from the group consisting of I or V;    -   (k) X₁₁ is selected from the group consisting of Y or F; and    -   (l) X₁₂ is selected from the group consisting of T, S, Q, N, or        A.

In a preferred embodiment, (a) X₁ is R; (b) X₂ is E; (c) X₃ is Q; (d) X₄is K; (e) X₅ is Y; (f) X₆ is D; (g) X₇ is L or W; (h) X₈ is P; (i) X₉ isL; (j) X₁₀ is I; (k) X₁₁ is Y; and (1) X₁₂ is Q or N.

In yet a further preferred embodiment, (a) X₁ is R; (b) X₂ is E; (c) X₃is Q; (d) X₄ is K; (e) X₅ is Y; (f) X₆ is D; (g) X₇ is L; (h) X₈ is P;(i) X₉ is L; (j) X₁₀ is I; (k) X₁₁ is Y; and (1) X₁₂ is Q.

In yet a further preferred embodiment, (a) X₁ is R; (b) X₂ is E; (c) X₃is Q; (d) X₄ is K; (e) X₅ is Y; (f) X₆ is D; (g) X₇ is W; (h) X₈ is P;(i) X₉ is L; (j) X₁₀ is I; (k) X₁₁ is Y; and (1) X₁₂ is N.

In certain embodiments, the CD loop comprises an amino acid sequenceselected from the group consisting of SEQ ID NOs: 101-125. In apreferred embodiment, the CD loop comprises the amino acid sequence setforth in SEQ ID NO: 106 or 113.

In certain embodiments, the invention provides a polypeptide comprisinga ¹⁰Fn3 domain comprising (i) a CD loop comprising an amino acidsequence having the consensus sequence of SEQ ID NO: 170 or the aminoacid sequence of any one of SEQ ID NOs: 101-125 and (ii) an amino acidsequence at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to thenon-CD loop regions of SEQ ID NOs: 23-100, 184-209 and 235-260 or thatdiffers from one of SEQ ID NOs: 23-100, 184-209 and 235-260 in at most1, 1-2, 1-5, 1-10 or 1-20 amino acids. In certain embodiments, thepolypeptide comprises an amino acid sequence that is at least 80%, 85%,90%, 95%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 23-100,184-209 and 235-260 or that differs from one of SEQ ID NOs: 23-100,184-209 and 235-260 in at most 1, 1-2, 1-5, 1-10 or 1-20 amino acids.Amino acid differences may be substitutions, additions or deletions.

In certain aspects, the invention provides a fusion polypeptidecomprising a fibronectin type III tenth (¹⁰Fn3) domain and aheterologous protein, wherein the ¹⁰Fn3 domain comprises a) AB, BC, CD,DE, EF, and FG loops, b) a CD loop with an altered amino acid sequencerelative to the sequence of the corresponding loop of the human ¹⁰Fn3domain, and c) wherein the polypeptide binds to human serum albumin witha K_(D) of less than 500 nM.

In certain embodiments, the fusion polypeptide comprises an albuminbinding Adnectin comprising an amino acid sequence at least 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one ofSEQ ID NOs: 23-100, 184-209 and 235-260 or that differs from one of SEQID NOs: 23-100, 184-209 and 235-260 in at most 1, 1-2, 1-5, 1-10 or 1-20amino acids. In a preferred embodiment, the fusion polypeptide comprisesan albumin binding Adnectin comprising the amino acid sequence of SEQ IDNO: 55, 81, 190 or 241. In yet another preferred embodiment, the fusionpolypeptide comprises an albumin binding Adnectin comprising the aminoacid sequence of SEQ ID NO: 62, 88, 197 or 248.

In certain embodiments, the fusion polypeptide comprises an albuminbinding Adnectin and a heterologous moiety, wherein the heterologousmoiety is a therapeutic moiety.

In certain embodiments, the heterologous protein is a polypeptidecomprising a ¹⁰Fn3 domain. In some embodiments, the ¹⁰Fn3 domain bindsto a target protein other than serum albumin. In one embodiment, the¹⁰Fn3 domain binds to PCSK9 (i.e., a PCSK9 Adnectin), and comprises anamino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to SEQ ID NO: 167 or that differs from SEQ IDNO: 167 in at most 1, 1-2, 1-5, 1-10 or 1-20 amino acids.

In certain embodiments, the fusion polypeptide is a PCSK9-PKE2 tandemAdnectin comprising an amino acid sequence at least 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 168, 169 or261 or that differs from one of SEQ ID NOs: 168, 169 or 261 in at most1, 1-2, 1-5, 1-10 or 1-20 amino acids (and may or may not comprise anN-terminal methionine).

In certain embodiments, the serum half-life of the fusion polypeptide inthe presence of mouse serum albumin is at least 10 hours. In someembodiments, the serum half-life of the fusion polypeptide in thepresence of cynomolgus serum albumin is at least 50 hours. In certainembodiments, the serum half-life of the fusion polypeptide in thepresence of mouse or cynomolgus serum albumin is 10-100 hours, such as10-90 hours, 10-80 hours, 10-70 hours, 10-60 hours, 10-50 hours, 10-40hours, 10-30 hours, 10-20 hours, 50-100 hours, 60-100 hours, 70-100hours, 80-100 hours, 90-100 hours, 20-90 hours, 30-80 hours, 40-70hours, or 50-60 hours.

In certain aspects, the invention provides a PKE2 Adnectin or PCSK9-PKE2tandem Adnectin comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 23-100, 168, 169, 184-209, 235-260, and261.

In certain aspects, the invention provides a composition comprising anyone of the albumin binding Adnectins or fusion proteins comprising such,as described herein, and a carrier.

In certain aspects, the invention provides an isolated nucleic acidmolecule encoding any one of the ablumin binding Adnectins or fusionproteins comprising such, as described herein, for example, those setforth in SEQ ID NOs: 126-151 and 172, expression vectors encoding thenucleic acid molecules, and cells comprising the nucleic acid molecules.Also provided are nucleic acids comprising a nucleotide sequence that isat least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical toany of these nucleotide sequences described herein, or which differtherefrom in at most 1-5, 1-10, 1-50 or 1-100 nucleotides.

In certain aspects, the invention provides a method of producing thealbumin binding Adnectins or fusion proteins comprising such describedherein, comprising culturing the cell comprising the nucleic acidmolecules encoding the same under conditions suitable for expressing theAdnectins or fusion proteins, and purifying the same.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic diagram of the competitive alpha-screen assaydescribed in Example 6.

FIG. 2 is a graph depicting the competition of various Adnectins withhuman FcRn receptor for binding to human serum albumin (HSA).

FIG. 3 is a graph depicting the plasma half-lives of 2629_E06 and2630_D02 PKE2 Adnectins in WT mice.

FIG. 4 is a graph depicting T-cell proliferation results for percentageantigenicity and strength of proliferative responses for 2629_E06 and2630_D02 Adnectins, and the parent 2270_C01 molecule.

FIG. 5 depicts a comparison of the modularity of tandem Adnectins. TheAdnectin 1318_H04 corresponds to a north pole-based serum albuminbinding Adnectin. The “X” refers to the configuration of non-PKE targetspecific Adnectin (i.e., myostatin; “myo”, or PCSK9). The lower paneldepicts a legend for the shades of grey in each box which correspond toHSA binding EC₅₀ tandem:monoAdnectin ratios as determined by directbinding ELISA (i.e., the darker the shade of grey, the stronger thebinding to HSA).

FIG. 6 shows the Bio-Layer Interferometry sensograms of PCSK9-PKE2tandem Adnectins binding to hPCSK9 in the presence or absence of HSA.

FIG. 7 is a Biacore sensogram showing the binding of the 4472_C06PCSK9-PKE2 tandem Adnectin first to HSA, then to PCSK9 after injectionof the corresponding proteins.

FIG. 8 is a graph depicting the in vivo PK profile of the tandemPCSK9-PKE2 Adnectin 4772_C06 in wild-type C57 Bl/6 mice.

FIG. 9 shows free PCSK9 levels following dosing of vehicle or PCSK9-PKE2Adnectin 4472_C06 at 0.5 mg/kg or 2 mg/kg in hPCSK9 transgenic mice.

FIG. 10 is a graph showing the plasma PK profiles and half-lives of PKE2Adnectin 2629_E06, PCSK9-PKE2 tandem 5190_E01 Adnectin, and PEGylatedPCSK9 in cynomolgus monkeys.

FIG. 11 is a graph showing the plasma half-life of PKE2 Adnectin2270_C01 in cynomolgus monkeys.

FIG. 12 is a graph showing the pharmacodynamic profile of LDL-c andPCSK9 in cynos following administration of the PCSK9-PKE2 tandemAdnectin 5190_E01 in cynomolgus monkeys. The profile demonstrates robustlowering of LDL-c, inhibition of free PCSK9 and an increase in totalPCSK9, all of which return to baseline by the end of the study.

FIG. 13 is a graph showing the LDL-c lowering effects of PCSK9-PKE2tandem Adnectin 5190_E01 and a PEGylated PCSK9 Adnectin comparator,along with the 2629_E06 PKE2 control in cynomolgus monkeys.

FIG. 14 shows target engagement by the tandem PCSK9-PKE2 Adnectin at twodifferent concentrations compared to PEGylated PCSK9 adnectin and PKE2Adnectin 2629_E06 in cynomolgus monkeys.

FIG. 15 shows total PCSK9 levels over time in cynomolgus monkeys afteradministration of the tandem PCSK9-PKE2 Adnectin, pegylated PCSK9adnectin or PKE2 Adnectin 2629_E06.

FIG. 16 is a graph depicting T-cell proliferation results for thepercent and strength of proliferative responses for the PCSK9-PKE2tandem Adnectins 4472_F08, 4472_E06, and 4472_C06, as well as thecomponent PKE2 Adnectin 2629_E06 and the component PCSK9 Adnectin2382_D09. The bars on the far left of the graph correspond to controlproteins with low, medium, and high antigenicity.

FIG. 17 shows the amino acid sequences of the PKE2 Adnectins describedherein.

FIGS. 18A-18C show the nucleic acid sequences of the PKE2 Adnectins andPCSK9-PKE2 tandem Adnectins described herein.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by the skilled artisan.Although any methods and compositions similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention, the preferred methods and compositions are described herein.

A “polypeptide,” as used herein, refers to any sequence of two or moreamino acids, regardless of length, post-translation modification, orfunction. “Polypeptide,” “peptide,” and “protein” are usedinterchangeably herein. Polypeptides can include natural amino acids andnon-natural amino acids such as those described in U.S. Pat. No.6,559,126, incorporated herein by reference. Polypeptides can also bemodified in any of a variety of standard chemical ways (e.g., an aminoacid can be modified with a protecting group; the carboxy-terminal aminoacid can be made into a terminal amide group; the amino-terminal residuecan be modified with groups to, e.g., enhance lipophilicity; or thepolypeptide can be chemically glycosylated or otherwise modified toincrease stability or in vivo half-life). Polypeptide modifications caninclude the attachment of another structure such as a cyclic compound orother molecule to the polypeptide and can also include polypeptides thatcontain one or more amino acids in an altered configuration (i.e., R orS; or, L or D).

A “polypeptide chain”, as used herein, refers to a polypeptide whereineach of the domains thereof is joined to other domain(s) by peptidebond(s), as opposed to non-covalent interactions or disulfide bonds.

An “isolated” polypeptide is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the polypeptide,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the polypeptide willbe purified (1) to greater than 95% by weight of polypeptide asdetermined by the Lowry method, and most preferably more than 99% byweight, (2) to a degree sufficient to obtain at least residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornonreducing condition using Coomassie blue or, preferably, silver stain.Isolated polypeptide includes the polypeptide in situ within recombinantcells since at least one component of the polypeptide's naturalenvironment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

A “region” of a ¹⁰Fn3 domain as used herein refers to either a loop (AB,BC, CD, DE, EF and FG), a β-strand (A, B, C, D, E, F and G), theN-terminus (corresponding to amino acid residues 1-7 of SEQ ID NO: 1),or the C-terminus (corresponding to amino acid residues 93-94 of SEQ IDNO: 1) of the human ¹⁰Fn3 domain.

A “north pole loop” refers to any one of the BC, DE and FG loops of afibronectin human fibronectin type 3 tenth (¹⁰Fn3) domain.

A “south pole loop” refers to any one of the AB, CD and EF loops of afibronectin human fibronectin type 3 tenth (¹⁰Fn3) domain.

A “scaffold region” refers to any non-loop region of a human ¹⁰Fn3domain. The scaffold region includes the A, B, C, D, E, F and Gβ-strands as well as the N-terminal region (amino acids corresponding toresidues 1-8 of SEQ ID NO: 1) and the C-terminal region (amino acidscorresponding to residues 93-94 of SEQ ID NO: 1 and optionallycomprising the 7 amino acids constituting the natural linker between the10^(th) and the 11^(th) repeat of the Fn3 domain in human fibronectin).

“Percent (%) amino acid sequence identity” herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in a selected sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared. For purposes herein,however, % amino acid sequence identity values are obtained as describedbelow by using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc. has been filed with user documentation in the U.S. CopyrightOffice, Washington D.C., 20559, where it is registered under U.S.Copyright Registration No. TXU510087, and is publicly available throughGenentech, Inc., South San Francisco, Calif. The ALIGN-2 program shouldbe compiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

For purposes herein, the % amino acid sequence identity of a given aminoacid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows: 100times the fraction X/Y where X is the number of amino acid residuesscored as identical matches by the sequence alignment program ALIGN-2 inthat program's alignment of A and B, and where Y is the total number ofamino acid residues in B. It will be appreciated that where the lengthof amino acid sequence A is not equal to the length of amino acidsequence B, the % amino acid sequence identity of A to B will not equalthe % amino acid sequence identity of B to A.

The terms “specifically binds,” “specific binding,” “selective binding,”and “selectively binds,” as used interchangeably herein refers to anAdnectin that exhibits affinity for a serum albumin, but does notsignificantly bind (e.g., less than about 10% binding) to a differentpolypeptide as measured by a technique available in the art such as, butnot limited to, Scatchard analysis and/or competitive binding assays(e.g., competition ELISA, BIACORE assay). The term is also applicablewhere e.g., a binding domain of an Adnectin of the invention is specificfor serum albumin.

The “half-life” of a polypeptide can generally be defined as the timetaken for the serum concentration of the polypeptide to be reduced by50%, in vivo, for example due to degradation of the polypeptide and/orclearance or sequestration of the polypeptide by natural mechanisms. Thehalf-life can be determined in any manner known per se, such as bypharmacokinetic analysis. Suitable techniques will be clear to theperson skilled in the art, and may, for example, generally involve thesteps of administering a suitable dose of a polypeptide to a primate;collecting blood samples or other samples from said primate at regularintervals; determining the level or concentration of the polypeptide insaid blood sample; and calculating, from (a plot of) the data thusobtained, the time until the level or concentration of the polypeptidehas been reduced by 50% compared to the initial level upon dosing.Methods for determining half-life may be found, for example, in Kennethet al., Chemical Stability of Pharmaceuticals: A Handbook forPharmacists (1986); Peters et al, Pharmacokinetic analysis: A PracticalApproach (1996); and “Pharmacokinetics”, M Gibaldi & D Perron, publishedby Marcel Dekker, 2nd Rev. edition (1982).

Half-life can be expressed using parameters such as the t_(1/2)-alpha,t_(1/2)-beta and the area under the curve (AUC). In the presentspecification, an “increase in half-life” refers to an increase in anyone of these parameters, any two of these parameters, or in all threethese parameters. In certain embodiments, an increase in half-liferefers to an increase in the t½-beta, either with or without an increasein the t_(1/2)-alpha and/or the AUC or both.

The term “K_(D),” as used herein, is intended to refer to thedissociation equilibrium constant of a particular Adnectin-proteininteraction or the affinity of an Adnectin for a protein (e.g., serumalbumin), as measured using a surface plasmon resonance assay or a cellbinding assay. A “desired K_(D),” as used herein, refers to a K_(D) ofan Adnectin that is sufficient for the purposes contemplated. Forexample, a desired K_(D) may refer to the K_(D) of an Adnectin requiredto elicit a functional effect in an in vitro assay, e.g., a cell-basedluciferase assay.

The term “k_(ass)”, as used herein, is intended to refer to theassociation rate constant for the association of an Adnectin into theAdnectin/protein complex.

The term “k_(diss)”, as used herein, is intended to refer to thedissociation rate constant for the dissociation of an Adnectin from theAdnectin/protein complex.

The term “IC₅₀”, as used herein, refers to the concentration of anAdnectin that inhibits a response, either in an in vitro or an in vivoassay, to a level that is 50% of the maximal inhibitory response, i.e.,halfway between the maximal inhibitory response and the untreatedresponse.

The term “therapeutically effective amount” refers to an amount of adrug effective to treat a disease or disorder in a mammal and/or relieveto some extent one or more of the symptoms associated with the disorder.

As used herein, “preventing” a disease or disorder refers to reducingthe probability of occurrence of a disease-state in a statistical samplerelative to an untreated control sample, or delaying the onset orreducing the severity of one or more symptoms of the disease or disorderrelative to the untreated control sample. Patients may be selected forpreventative therapy based on factors that are known to increase risk ofsuffering a clinical disease state compared to the general population.The term “treating” as used herein includes (a) inhibiting thedisease-state, i.e., arresting its development; and/or (b) relieving thedisease-state, i.e., causing regression of the disease state once it hasbeen established.

Overview

The novel fibronectin based scaffold polypeptides described herein bindto serum albumin of various species and can be coupled to additionalmolecule(s), such as other ¹⁰Fn3 domains that bind to different targets,or polypeptides for which increased half-life is beneficial.

A. General Structure of Fibronectin Based Scaffolds

Fn3 refers to a type III domain from fibronectin. An Fn3 domain issmall, monomeric, soluble, and stable. It lacks disulfide bonds and,therefore, is stable under reducing conditions. The overall structure ofFn3 resembles the immunoglobulin fold. Fn3 domains comprise, in orderfrom N-terminus to C-terminus, a beta or beta-like strand, A; a loop,AB; a beta or beta-like strand, B; a loop, BC; a beta or beta-likestrand, C; a loop, CD; a beta or beta-like strand, D; a loop, DE; a betaor beta-like strand, E; a loop, EF; a beta or beta-like strand, F; aloop, FG; and a beta or beta-like strand, G. The seven antiparallelβ-strands are arranged as two beta sheets that form a stable core, whilecreating two “faces” composed of the loops that connect the beta orbeta-like strands. Loops AB, CD, and EF are located at one face (“thesouth pole”) and loops BC, DE, and FG are located on the opposing face(“the north pole”). Any or all of loops AB, BC, CD, DE, EF and FG mayparticipate in ligand binding. There are at least 15 different Fn3modules in human Fibronectin, and while the sequence homology betweenthe modules is low, they all share a high similarity in tertiarystructure.

In some embodiments, the Fn3 domain is an Fn3 domain derived from thewild-type tenth module of the human fibronectin type III domain (¹⁰Fn3):

(SEQ ID NO: 1) VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT(AB, CD, and EF loops are underlined).

In some embodiments, the non-ligand binding sequences of ¹⁰Fn3, i.e.,the “¹⁰Fn3 scaffold”, may be altered provided that the ¹⁰Fn3 retainsligand binding function and/or structural stability. A variety of mutant¹⁰Fn3 scaffolds have been reported. In one aspect, one or more of Asp 7,Glu 9, and Asp 23 is replaced by another amino acid, such as, forexample, a non-negatively charged amino acid residue (e.g., Asn, Lys,etc.). These mutations have been reported to have the effect ofpromoting greater stability of the mutant ¹⁰Fn3 at neutral pH ascompared to the wild-type form (see, e.g., PCT Publication No. WO02/04523). A variety of additional alterations in the ¹⁰Fn3 scaffoldthat are either beneficial or neutral have been disclosed. See, forexample, Batori et al., Protein Eng., 15(12):1015-1020 (December 2002);Koide et al., Biochemistry, 40(34):10326-10333 (Aug. 28, 2001).

Both variant and wild-type ¹⁰Fn3 proteins are characterized by the samestructure, namely seven beta-strand domain sequences designated Athrough G and six loop regions (AB loop, BC loop, CD loop, DE loop, EFloop, and FG loop) which connect the seven beta-strand domain sequences.The beta strands positioned closest to the N- and C-termini may adopt abeta-like conformation in solution. In SEQ ID NO: 1, the AB loopcorresponds to residues 14-17, the BC loop corresponds to residues23-31, the CD loop corresponds to residues 37-47, the DE loopcorresponds to residues 51-56, the EF loop corresponds to residues63-67, and the FG loop corresponds to residues 76-87.

Accordingly, in some embodiments, the serum albumin binding Adnectin ofthe invention is an ¹⁰Fn3 polypeptide that is at least 40%, 50%, 60%,65%, 70%, 75%, 80%, 85%, or 90% identical to the human ¹⁰Fn3 domain,shown in SEQ ID NO: 1. Much of the variability will generally occur inone or more of the loops. Each of the beta or beta-like strands of a¹⁰Fn3 polypeptide may consist essentially of an amino acid sequence thatis at least 80%, 85%, 90%, 95% or 100% identical to the sequence of acorresponding beta or beta-like strand of SEQ ID NO: 1, provided thatsuch variation does not disrupt the stability of the polypeptide inphysiological conditions.

Additionally, insertions and deletions in the loop regions may also bemade while still producing high affinity serum-binding ¹⁰Fn3 bindingdomains. Accordingly, in some embodiments, one or more loops selectedfrom AB, BC, CD, DE, EF and FG may be extended or shortened in lengthrelative to the corresponding loop in wild-type human ¹⁰Fn3. In anygiven polypeptide, one or more loops may be extended in length, one ormore loops may be reduced in length, or combinations thereof. In someembodiments, the length of a given loop may be extended by 2-25, 2-20,2-15, 2-10, 2-5, 5-25, 5-20, 5-15, 5-10, 10-25, 10-20, or 10-15 aminoacids. In some embodiments, the length of a given loop may be reduced by1-15, 1-11, 1-10, 1-5, 1-3, 1-2, 2-10, or 2-5 amino acids.

As described above, amino acid residues corresponding to residues 14-17,23-30, 37-47, 51-56, 63-67 and 76-87 of SEQ ID NO: 1 define the AB, BC,CD, DE, EF and FG loops, respectively. However, it should be understoodthat not every residue within a loop region needs to be modified inorder to achieve a ¹⁰Fn3 binding domain having strong affinity for adesired target. In some embodiments, only residues in a loop, e.g., theCD loop are modified to produce high affinity target binding ¹⁰Fn3domains.

In some embodiments, the invention provides polypeptides comprising a¹⁰Fn3 domain, wherein the ¹⁰Fn3 domain comprises AB, BC, CD, DE, and FGloops, and has at least one loop selected from AB, CD, and EF loops withan altered amino acid sequence relative to the sequence of thecorresponding loop of the human ¹⁰Fn3 domain of SEQ ID NO: 1. In someembodiments, the AB, CD, and EF loops are altered. In certainembodiments, only the AB loop is altered. In certain embodiments, onlythe CD loop is altered. In certain embodiments, only the EF loop isaltered. In certain embodiments, the AB and CD loops are both altered.In certain embodiments, the AB and EF loops are both altered. In certainembodiments, the CD and EF loops are both altered. In some embodiments,one or more specific scaffold alterations are combined with one or moreloop alterations. By “altered” is meant one or more amino acid sequencealterations relative to a template sequence (i.e., the correspondingwild-type human fibronectin domain) and includes amino acid additions,deletions, and substitutions.

In some embodiments, the fibronectin based scaffold protein comprises a¹⁰Fn3 domain having a combination of north and south pole loopalterations. For example, one or more of loops AB, CD, and EF, incombination with one or more of loops BC, DE, and FG, can be alteredrelative to the corresponding loops of the human ¹⁰Fn3 domain of SEQ IDNO: 1.

In some embodiments, the polypeptide comprises a ¹⁰Fn3 domain thatcomprises an amino acid sequence at least 80, 85, 90, 95, 98, 99, or100% identical to the non-loop regions and/or non-modified loop regionsof SEQ ID NO: 1, wherein at least one loop selected from AB, CD, and EFis altered. For example, in certain embodiments, the AB loop may have upto 4 amino acid substitutions, up to 10 amino acid insertions, up to 3amino acid deletions, or a combination thereof; the CD loop may have upto 6 amino acid substitutions, up to 10 amino acid insertions, up to 4amino acid deletions, or a combination thereof; and the EF loop may haveup to 5 amino acid substations, up to 10 amino acid insertions, up to 3amino acid deletions, or a combination thereof; and/or the FG loop mayhave up to 12 amino acid substitutions, up to 11 amino acid deletions,up to 25 amino acid insertions, or a combination thereof.

In some embodiments, one or more residues of the integrin-binding motif“arginine-glycine-aspartic acid” (RGD) (amino acids 78-80 of SEQ IDNO: 1) may be substituted so as to disrupt integrin binding. In someembodiments, the FG loop of the polypeptides provided herein does notcontain an RGD integrin binding site. In one embodiment, the RGDsequence is replaced by a polar amino acid-neutral amino acid-acidicamino acid sequence (in the N-terminal to C-terminal direction). Incertain embodiments, the RGD sequence is replaced with SGE. In yetcertain embodiments, the RGD sequence is replaced with RGE.

In certain embodiments, the fibronectin based scaffold protein comprisesa ¹⁰Fn3 domain that is defined generally by following the sequence:

(SEQ ID NO: 2) VSDVPRDLEVVAA(X)_(u) LLISW(X)_(v) YRITY(X)_(w) FTV(X)_(x)ATISGL (X)_(y) YTITVYA(X)_(z) ISINYRT

In SEQ ID NO: 2, the AB loop is represented by (X)_(u), the BC loop isrepresented by (X)_(v), the CD loop is represented by (X)_(w), the DEloop is represented by (X)_(x), the EF loop is represented by (X)_(y)and the FG loop is represented by X_(z). X represents any amino acid andthe subscript following the X represents an integer of the number ofamino acids. In particular, u, v, w, x, y and z may each independentlybe anywhere from 2-20, 2-15, 2-10, 2-8, 5-20, 5-15, 5-10, 5-8, 6-20,6-15, 6-10, 6-8, 2-7, 5-7, or 6-7 amino acids. The sequences of the betastrands (underlined) may have anywhere from 0 to 10, from 0 to 8, from 0to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1substitutions, deletions or additions across all 7 scaffold regionsrelative to the corresponding amino acids shown in SEQ ID NO: 2. In someembodiments, the sequences of the beta strands may have anywhere from 0to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3,from 0 to 2, or from 0 to 1 conservative substitutions across all 7scaffold regions relative to the corresponding amino acids shown in SEQID NO: 2. In certain embodiments, the hydrophobic core amino acidresidues (bolded residues in SEQ ID NO: 2 above) are fixed, and anysubstitutions, conservative substitutions, deletions or additions occurat residues other than the hydrophobic core amino acid residues. In someembodiments, the hydrophobic core residues of the polypeptides providedherein have not been modified relative to the wild-type human ¹⁰Fn3domain (SEQ ID NO: 1).

In some embodiments, the amino acid sequences of the N-terminal and/orC-terminal regions of the polypeptides provided herein may be modifiedby deletion, substitution or insertion relative to the amino acidsequences of the corresponding regions of the wild-type human ¹⁰Fn3domain (SEQ ID NO: 1). The ¹⁰Fn3 domains generally begin with amino acidnumber 1 of SEQ ID NO: 1. However, domains with amino acid deletions arealso encompassed by the invention. Additional sequences may also beadded to the N- or C-terminus of a ¹⁰Fn3 domain having the amino acidsequence of SEQ ID NO: 1. For example, in some embodiments, theN-terminal extension consists of an amino acid sequence selected fromthe group consisting of: M, MG, and G.

In exemplary embodiments, an alternative N-terminal region having from1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2, or 1 amino acids in lengthcan be added to the N-terminal region of SEQ ID NO: 1. Exemplaryalternative N-terminal regions include (represented by the single letteramino acid code) M, MG, G, MGVSDVPRDL (SEQ ID NO: 3) and GVSDVPRDL (SEQID NO: 4). Other suitable alternative N-terminal regions include, forexample, X_(n)SDVPRDL (SEQ ID NO: 5), X_(n)DVPRDL (SEQ ID NO: 6),X_(n)VPRDL (SEQ ID NO: 7), X_(n)PRDL (SEQ ID NO: 8) X_(n)RDL (SEQ ID NO:9), X_(n)DL (SEQ ID NO: 10), or X_(n)L, wherein n=0, 1 or 2 amino acids,wherein when n=1, X is Met or Gly, and when n=2, X is Met-Gly. When aMet-Gly sequence is added to the N-terminus of a ¹⁰Fn3 domain, the Mwill usually be cleaved off, leaving a G at the N-terminus. In certainembodiments, the alternative N-terminal region comprises the amino acidsequence MASTSG (SEQ ID NO: 11).

In exemplary embodiments, an alternative C-terminal region having from1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2, or 1 amino acids in lengthcan be added to the C-terminal region of SEQ ID NO: 1. Specific examplesof alternative C-terminal region sequences include, for example,polypeptides comprising, consisting essentially of, or consisting of,EIEK (SEQ ID NO: 12), EGSGC (SEQ ID NO: 13), EIEKPCQ (SEQ ID NO: 14),EIEKPSQ (SEQ ID NO: 15), EIEKP (SEQ ID NO: 16), EIEKPS (SEQ ID NO: 17),or EIEKPC (SEQ ID NO: 18). In some embodiments, the alternativeC-terminal region comprises EIDK (SEQ ID NO: 19), and in particularembodiments, the alternative C-terminal region is either EIDKPCQ (SEQ IDNO: 20) or EIDKPSQ (SEQ ID NO: 21). Additional suitable alternativeC-terminal regions include those set forth in Table 20 and SEQ ID NOs:210-220.

In certain embodiments, the C-terminal extension sequences comprise Eand D residues, and may be between 8 and 50, 10 and 30, 10 and 20, 5 and10, and 2 and 4 amino acids in length. In some embodiments, tailsequences include ED-based linkers in which the sequence comprisestandem repeats of ED. In exemplary embodiments, the tail sequencecomprises 2-10, 2-7, 2-5, 3-10, 3-7, 3-5, 3, 4 or 5 ED repeats. Incertain embodiments, the ED-based tail sequences may also includeadditional amino acid residues, such as, for example: EI, EID, ES, EC,EGS, and EGC. Such sequences are based, in part, on known Adnectin tailsequences, such as EIDKPSQ (SEQ ID NO: 21), in which residues D and Khave been removed. In exemplary embodiments, the ED-based tail comprisesan E, I or EI residues before the ED repeats.

In certain embodiments, an alternative C-terminal moiety, which can belinked to the C-terminal amino acids RT (i.e., amino acid 93-94 of SEQID NO: 1) of any of the Adnectins provided herein comprises the aminoacids P_(m)X_(n), wherein P is proline, X is any amino acid, m is aninteger that is at least 1 and n is 0 or an interger that is at least 1.In certain embodiments, the alternative C-terminal moiety comprises theamino acids PC. In certain embodiments, the alternative C-terminalmoiety comprises the amino acids PI, PC, PID, PIE, PIDK (SEQ ID NO:221), PIEK (SEQ ID NO: 222), PIDKP (SEQ ID NO: 223), PIEKP (SEQ ID NO:224), PIDKPS (SEQ ID NO: 225), PIEKPS (SEQ ID NO: 226), PIDKPC (SEQ IDNO: 227), PIEKPC (SEQ ID NO: 228), PIDKPSQ (SEQ ID NO: 229), PIEKPSQ(SEQ ID NO: 230), PIDKPCQ (SEQ ID NO: 231), PIEKPCQ (SEQ ID NO: 232),PHHHHHH (SEQ ID NO: 233), and PCHHHHHH (SEQ ID NO: 234).

In certain embodiments, the fibronectin based scaffold proteins comprisea ¹⁰Fn3 domain having both an alternative N-terminal region sequence andan alternative C-terminal region sequence.

B. Serum Albumin Binders Having Modified South Pole Loop(s)

¹⁰Fn3 domains are cleared rapidly from circulation via renal filtrationand degradation due to their small size of about 10 kDa (t_(1/2)=15-45minutes in mice; 3 hours in monkeys). In certain aspects, theapplication provides ¹⁰Fn3 domains with south pole modifications thatbind specifically to serum albumin, e.g., human serum albumin (HSA) toprolong the t_(1/2) of the ¹⁰Fn3 domain.

HSA has a serum concentration of 600 μM and a t_(1/2) of 19 days inhumans. The extended t_(1/2) of HSA has been attributed, in part, to itsrecycling via the neonatal Fc receptor (FcRn). HSA binds FcRn in apH-dependent manner after endosomal uptake into endothelial cells; thisinteraction recycles HSA back into the bloodstream, thereby shunting itaway from lysosomal degradation. FcRn is widely expressed and therecycling pathway is thought to be constitutive. In the majority of celltypes, most FcRn resides in the intracellular sorting endosome. HSA isreadily internalized by a nonspecific mechanism of fluid-phasepinocytosis and rescued from degradation in the lysosome by FcRn. At theacidic pH found in the endosome, HSA's affinity for FcRn increases (5 μMat pH 6.0). Once bound to FcRn, HSA is shunted away from the lysosomaldegradation pathway, transcytosed to and released at the cell surface.

North pole-based serum albumin binding Adnectins, herein referred to as“first generation” serum albumin binding Adnectins, have been describedin, e.g., WO2011140086. In order to improve upon first generation northpole-based serum albumin binding Adnectins (SABAs), of which some didnot bind to mouse or rat serum albumin, did not have high affinity forserum albumins across species, and were not always compatible in amultivalent ¹⁰Fn3-based platform, second generation south pole-basedserum albumin binding Adnectins (PKE2 Adnectins) with modified southpole loops were developed as described in the Examples.

Accordingly, in one aspect, the invention provides a ¹⁰Fn3 domain having(i) a modification in the amino acid sequence of at least one south poleloop selected from the AB, CD, and EF loops relative to thecorresponding loop of the wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1),wherein the ¹⁰Fn3 domain binds to serum albumin (e.g., human serumalbumin). The modified south pole loop(s) contribute to binding to thesame target. Various combinations of modified south pole loops arecontemplated. For example, a ¹⁰Fn3 may comprise one modified south poleloops, two modified south pole loops, or even all three south pole loopsmodified. In certain embodiments, one or more modified south pole loopscan be made in conjunction with one or more modified north pole loops(i.e., one or more of BC, DE, and FG loops). The modified loops may havesequence modifications across an entire loop or only in a portion of theloop. Additionally, one or more of the modified loops may haveinsertions or deletions such that the length of the loop is variedrelative to the length of the corresponding loop of the wild-typesequence (i.e., SEQ ID NO: 1). In certain embodiments, additionalregions in the ¹⁰Fn3 domain (i.e., in addition to the south pole loops),such as 3-strand, N-terminal and/or C-terminal regions, may also bemodified in sequence relative to the wild-type ¹⁰Fn3 domain, and suchadditional modifications may also contribute to binding to the target.In certain embodiments, a South Pole loop is the only domain that ismodified. In specific embodiments, the CD loop is the only domain thatis modified. In certain embodiments, the serum binding ¹⁰Fn3 domain maybe modified to comprise an N-terminal extension sequence and/or aC-terminal extension sequence, as described supra.

In one embodiment, the invention provides Adnectins that bind to serumalbumin having an altered CD loop relative to the corresponding loop ofthe wild-type human ¹⁰Fn3 domain, for example, ¹⁰Fn3 domains set forthin SEQ ID NOs: 23-100, 184-209 and 235-260. In some embodiments, thealbumin binding Adnectins comprise, or alternatively lack a 6× his tail.In some embodiments, the albumin binding Adnectins correspond to coreAdnectins which lack the N-terminal leader and C-terminal tail, as setforth in SEQ ID NOs: 75-100.

In exemplary embodiments, the serum albumin binding ¹⁰Fn3 proteinsdescribed herein bind to human serum albumin with a K_(D) of less than 3μM, 2.5 μM, 2 μM, 1.5 μM, 1 μM, 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500μM, 100 μM, 100 μM, 50 μM, or 10 μM. The Kd may be, e.g., in the rangeof 0.1 nM to 50 nM, 0.1 nM to 100 nM, 0.1 nM to 1 μM, 0.5 nM to 50 nM,0.5 nM to 100 nM, 0.5 nM to 1 μM, 1 nM to 50 nM, 1 nM to 100 nM or 1 nMto 1 μM.

In certain embodiments, the albumin binding Adnectins (or ¹⁰Fn3proteins) described herein may also bind serum albumin from one or moreof cynomolgus monkey, rhesus monkey, rat, or mouse.

In certain embodiments, the serum albumin binding ¹⁰Fn3 proteinsdescribed herein bind to rhesus serum albumin (RhSA) or cynomolgousmonkey serum albumin (CySA) with a K_(D) of less than 3 μM, 2.5 μM, 2μM, 1.5 μM, 1 μM, 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 μM or 100 μM.The K_(D) may be, e.g., in the range of 0.1 nM to 50 nM, 0.1 nM to 100nM, 0.1 nM to 1 μM, 0.5 nM to 50 nM, 0.5 nM to 100 nM, 0.5 nM to 1 μM, 1nM to 50 nM, 1 nM to 100 nM or 1 nM to 1 μM.

In certain embodiments, the serum albumin binding ¹⁰Fn3 proteinsdescribed herein bind to rhesus serum albumin (RhSA), cynomolgous monkeyserum albumin (CySA), and mouse serum albumin (MSA) with a K_(D) of lessthan 3 μM, 2.5 μM, 2 μM, 1.5 μM, 1 μM, 500 nM, 100 nM, 50 nM, 10 nM, 1nM, 500 μM or 100 μM. The K_(D) may be, e.g., in the range of 0.1 nM to50 nM, 0.1 nM to 100 nM, 0.1 nM to 1 μM, 0.5 nM to 50 nM, 0.5 nM to 100nM, 0.5 nM to 1 μM, 1 nM to 50 nM, 1 nM to 100 nM or 1 nM to 1 μM.

In certain embodiments, the serum albumin binding ¹⁰Fn3 proteinsdescribed herein bind to rhesus serum albumin (RhSA), cynomolgous monkeyserum albumin (CySA), mouse serum albumin (MSA), and rat serum albumin(RSA) with a K_(D) of less than 3 μM, 2.5 μM, 2 M, 1.5 μM, 1 μM, 500 nM,100 nM, 50 nM, 10 nM, 1 nM, 500 μM or 100 μM. The K_(D) may be, e.g., inthe range of 0.1 nM to 50 nM, 0.1 nM to 100 nM, 0.1 nM to 1 μM, 0.5 nMto 50 nM, 0.5 nM to 100 nM, 0.5 nM to 1 μM, 1 nM to 50 nM, 1 nM to 100nM or 1 nM to 1 μM.

In certain embodiments, the albumin binding Adnectins described hereinbind to serum albumin at a pH range of 5.5 to 7.4.

In certain embodiments, the albumin binding Adnectins described hereinbind to domain I-II of human serum albumin.

In certain embodiments, the serum half-life of the albumin bindingAdnectins of the invention or the serum half-life of the albumin bindingAdnectins linked to a heterologous moiety, e.g., a second Adnectin, isat least 2 hours, 2.5 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30hours, 35 hours, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90hours, 100 hours, 110 hours, 120 hours, 130 hours, 135 hours, 140 hours,150 hours, 160 hours, or 200 hours. In certain embodiments, the serumhalf-life of the albumin binding Adnectins or the serum half-life of thealbumin binding Adnectins linked to a heterologous moiety, e.g., asecond Adnectin, is 2-200 hours, 5-200 hours, 10-200 hours, 25-200hours, 50-200 hours, 100-200 hours, 150-200 hours, 2-150 hours, 2-100hours, 2-50 hours, 2-25 hours, 2-10 hours, 2-5 hours, 5-150 hours,10-100 hours, or 25-50 hours.

In certain embodiments, the albumin binding Adnectins comprises asequence having at least 40%, 50%, 60%, 70%, 75%, 80% or 85% identity tothe wild-type ¹⁰Fn3 domain (SEQ ID NO: 1). In one embodiment, at leastone of the AB, CD, or EF loops is modified relative to the wild-type¹⁰Fn3 domain. In certain embodiments, at least two of the AB, CD, or EFloops are modified relative to the wild-type ¹⁰Fn3 domain. In certainembodiments, all three of the AB, CD, or EF loops are modified relativeto the wild-type ¹⁰Fn3 domain. In certain embodiments, a serum albuminbinding ¹⁰Fn3 domain comprises a sequence having at least 40%, 50%, 60%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to anyone of SEQ ID NOs: 23-100, 184-209 and 235-260.

In certain embodiments, a serum albumin binding ¹⁰Fn3 domain (orAdnectin) may comprise the sequence as set forth in SEQ ID NO: 2,wherein the CD loop is represented by (X)_(w) and is replaced with a CDloop from any of the 26 core PKE2 Adnectin sequences (i.e., SEQ ID NOs:75-100). The scaffold regions of such albumin binding Adnectins may haveanywhere from 0 to 20, from 0 to 15, from 0 to 10, from 0 to 8, from 0to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1substitutions, conservative substitutions, deletions or additionsrelative to the scaffold amino acids residues of SEQ ID NO: 1. Suchscaffold modifications may be made, so long as the ablumin bindingAdnectin is capable of binding serum albumin, e.g., HSA, with a desiredK_(D).

In some embodiments, the CD loop region of the albumin binding Adnectinsof the invention can be described according to a consensus sequence.

Accordingly, in some embodiments, the CD loop is defined by theconsensus sequence

(SEQ ID NO: 170) G-X₁-X₂-V-X₃-X₄-X₅-S-X₆-X₇-G-X₈-X₉-Y-X₁₀-X₁₁-X₁₂-E,wherein,

-   -   (a) X₁ is selected from the group consisting of R or W;    -   (b) X₂ is selected from the group consisting of H, E, D, Y, or        Q;    -   (c) X₃ is selected from the group consisting of Q or H;    -   (d) X₄ is selected from the group consisting of I, K, M, Q, L,        or V;    -   (e) X₅ is selected from the group consisting of Y, F, or N;    -   (f) X₆ is selected from the group consisting of D, V, or E;    -   (g) X₇ is selected from the group consisting of L, W, or F;    -   (h) X₈ is selected from the group consisting of P or T;    -   (i) X₉ is selected from the group consisting of L or M;    -   (j) X₁₀ is selected from the group consisting of I or V;    -   (k) X₁₁ is selected from the group consisting of Y or F; and    -   (l) X₁₂ is selected from the group consisting of T, S, Q, N, or        A.

In certain preferred embodiments,

-   -   (a) X₁ is R;    -   (b) X₂ is E;    -   (c) X₃ is Q;    -   (d) X₄ is K;    -   (e) X₅ is Y;    -   (f) X₆ is D;    -   (g) X₇ is L or W;    -   (h) X₈ is P;    -   (i) X₉ is L;    -   (j) X₁₀ is I;    -   (k) X₁₁ is Y; and    -   (l) X₁₂ is Q or N.

In a preferred embodiment, X₇ is L and X₁₂ is Q.

In another preferred embodiment, X₇ is W and X₁₂ is N.

In some embodiments, the albumin binding Adnectins of the inventioncomprise a CD loop having sequences at least 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% identical to the CD loop sequences set forthin SEQ ID NOs: 101-125, or comprise at most 1, 1-2 or 1-3 amino aciddifference (i.e., substitution, e.g., deletion, addition or conservativesubstitution). The scaffold regions of such albumin binding Adnectinsmay comprise anywhere from 0 to 20, from 0 to 15, from 0 to 10, from 0to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2,or from 0 to 1 substitutions, conservative substitutions, deletions oradditions relative to the scaffold amino acids residues of SEQ ID NO: 1.Such scaffold modifications may be made, so long as the Adnectins arecapable of binding to serum albumin with a desired K_(D).

In a preferred embodiment, the CD loop of the albumin binding Adnectinsof the invention comprises an amino acid sequence selected from thegroup consisting of:

(SEQ ID NO: 101) GRHVQIYSDLGPLYIYTE, (SEQ ID NO: 102)GRHVHIYSDWGPMYIYTE, (SEQ ID NO: 103) GREVQKYSVLGPLYIYTE,(SEQ ID NO: 104) GREVQMYSDLGPLYVYSE, (SEQ ID NO: 105)GREVQKFSDWGPLYIYTE, (SEQ ID NO: 106) GREVQKYSDLGPLYIYQE,(SEQ ID NO: 107) GREVHQYSDWGPMYIYNE, (SEQ ID NO: 108)GREVHKNSDWGTLYIYTE, (SEQ ID NO: 109) GREVQKYSDLGPLYIYAE,(SEQ ID NO: 110) GREVHLYSDWGPMYIYTE, (SEQ ID NO: 111)GRHVQMYSDLGPLYIFSE, (SEQ ID NO: 112) GREVHMYSDFGPMYIYTE,(SEQ ID NO: 113) GREVQKYSDWGPLYIYNE, (SEQ ID NO: 114)GREVQMYSDLGPLYIYNE, (SEQ ID NO: 115) GREVQMYSDLGPLYIYTE,(SEQ ID NO: 116) GRHVQIYSDLGPLYIYNE, (SEQ ID NO: 117)GREVQIYSDWGPLYIYNE, (SEQ ID NO: 118) GREVQKYSDWGPLYIYQE,(SEQ ID NO: 119) GRHVHLYSEFGPMYIYNE, (SEQ ID NO: 120)GRDVHMYSDWGPMYIYQE, (SEQ ID NO: 121) GRHVQIYSDWGPLYIYNE,(SEQ ID NO: 122) GRYVQLYSDWGPMYIYTE, (SEQ ID NO: 123)GRQVQVFSDLGPLYIYNE, (SEQ ID NO: 124) GRQVQIYSDWGPLYIYNE, and(SEQ ID NO: 125) GRQVQMYSDWGPLYIYAE.

In some embodiments, the albumin binding Adnectin comprises an aminoacid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100%identical to any one of SEQ ID NOs: 23-100, 184-209 and 235-260 ordiffers therefrom in at most 1, 1-2, 1-3, 1-5, 1-10 or 1-20 amino aciddifferences, e.g., amino acid deletions, additions or substitutions(e.g., conservative substitutions). In certain embodiments, the albuminbinding molecules comprise an amino acid sequence at least 80%, 85%,90%, 95%, 98%, 99% or 100% identical to the non-CD loop region of SEQ IDNOs: 23-100, 184-209 and 235-260.

In a preferred embodiment, the albumin binding Adnectin comprises theamino acid sequence set forth in any one of SEQ ID NOs: 29, 55, 81, 190and 241. In another preferred embodiment, the albumin binding Adnectincomprises the amino acid sequence set forth in any one of SEQ ID NOs:36, 62, 88, 197 and 248.

In some embodiments, the invention provides mutant albumin bindingAdnectin molecules which have a cysteine residue introduced at aspecific position. Exemplary cysteine mutations are A12C, A26C, S55C,T56C and T58C (see Table 7 in the Examples). In a preferred embodiment,the cysteine mutations do not substantially alter the binding of thealbumin binding Adnectin to serum albumin.

In certain embodiments, a proline residue is introduced at theC-terminus of the ¹⁰Fn3 domain, for example, as shown, e.g., in SEQ IDNOs: 184-209 and 235-260. In certain embodiments, the proline residue isintroduced at the C-terminus of a tandem albumin binding Adnectin, asshown, e.g., in SEQ ID NO: 168 and 261. Addition of the proline residuedoes not preclude the addition of additional amino acid sequences to theC-termius of an albumin binding Adnectin or tandem albumin bindingAdnectin.

C. Cross-Competing Adnectins and/or Adnectins that Bind to the SameAdnectin Binding Site

Provided herein are proteins, such as Adnectins, antibodies or antigenbinding fragments thereof, small molecules, peptides, and the like thatcompete (e.g., cross-compete) for binding to serum albumin (e.g., HSA)with the particular PKE2 Adnectins described herein. Such competingproteins, e.g., Adnectins, can be identified based on their ability tocompetitively inhibit binding to serum albumin (e.g., HSA) of Adnectinsdescribed herein in standard serum albumin binding assays. For example,standard ELISA assays can be used in which a recombinant serum albuminprotein is immobilized on the plate, one of the proteins isfluorescently labeled and the ability of non-labeled protein to competeoff the binding of the labeled protein is evaluated.

The following exemplary competition assays are provided in the contextof Adnectins competing for binding to serum albumin with one of the PKE2proteins described herein. The same assays can be performed where anon-Adnectin protein is tested for competition. In one embodiment, acompetitive ELISA format can be performed to determine whether two serumalbumin Adnectins bind overlapping Adnectin binding sites (epitopes) onserum albumin (e.g., HSA). In one format, Adnectin #1 is coated on aplate, which is then blocked and washed. To this plate is added eitherserum albumin alone, or serum albumin pre-incubated with a saturatingconcentration of Adnectin #2. After a suitable incubation period, theplate is washed and probed with a polyclonal anti-serum albuminantibody, followed by detection with streptavidin-HRP conjugate andstandard tetramethylbenzidine development procedures. If the OD signalis the same with or without preincubation with Adnectin #2, then the twoAdnectins bind independently of one another, and their Adnectin bindingsites do not overlap. If, however, the OD signal for wells that receivedserum albumin/Adnectin#2 mixtures is lower than for those that receivedserum albumin alone, then binding of Adnectin #2 is confirmed to blockbinding of Adnectin #1 to serum albumin.

Alternatively, a similar experiment is conducted by surface plasmonresonance (SPR, e.g., BIAcore). Adnectin #1 is immobilized on an SPRchip surface, followed by injections of either serum albumin alone orserum albumin pre-incubated with a saturating concentration of Adnectin#2. If the binding signal for serum albumin/Adnectin#2 mixtures is thesame or higher than that of serum albumin alone, then the two Adnectinsbind independently of one another, and their Adnectin binding sites donot overlap. If, however, the binding signal for serumalbumin/Adnectin#2 mixtures is lower than the binding signal for serumalbumin alone, then binding of Adnectin #2 is confirmed to block bindingof Adnectin #1 to serum albumin. A feature of these experiments is theuse of saturating concentrations of Adnectin #2. If serum albumin is notsaturated with Adnectin #2, then the conclusions above do not hold.Similar experiments can be used to determine if any two serum albuminbinding proteins bind to overlapping Adnectin binding sites.

Both assays exemplified above may also be performed in the reverse orderwhere Adnectin#2 is immobilized and serum albumin—Adnectin#1 are addedto the plate. Alternatively, Adnectin #1 and/or #2 can be replaced witha monoclonal antibody and/or soluble receptor-Fc fusion protein.

In certain embodiments, competition can be determined using a HTRFsandwich assay.

In certain embodiments, the competing Adnectin is an Adnectin that bindsto the same Adnectin binding site on serum albumin as a particular PKE2Adnectin described herein. Standard mapping techniques, such as proteasemapping, mutational analysis, x-ray crystallography and 2-dimensionalnuclear magnetic resonance, can be used to determine whether an Adnectinbinds to the same Adnectin binding site as a reference Adnectin (see,e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol.66, G. E. Morris, Ed. (1996)).

Candidate competing albumin binding proteins, e.g., Adnectins, caninhibit the binding of PKE2 Adnectins of the invention to serum albumin(e.g., HSA) by at least 50%, at least 55%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 97%, at least 98%, or at least 99%. The %competition can be determined using the methods described above.

D. Multivalent/Tandem Adnectins

Provided herein are multivalent proteins that comprise two or more ¹⁰Fn3domains binding specifically to a target (Adnectins). For example, amultivalent protein may comprise 2, 3 or more ¹⁰Fn3 domains that arecovalently associated. In exemplary embodiments, multivalent protein isa bispecific or dimeric protein comprising two ¹⁰Fn3 domains. In certainembodiments, a multivalent protein comprises a first ¹⁰Fn3 domain thatbinds to serum albumin (e.g., human serum albumin) and a second ¹⁰Fn3domain that binds to a second target molecule (e.g., PCSK9). When boththe first and second target molecules are serum albumin, the first andsecond ¹⁰Fn3 domains may bind to the same or different epitopes.Additionally, when the first and second target molecules are the same,the regions of modification in the ¹⁰Fn3 domain that are associated withtarget binding may be the same or different. In exemplary embodiments,each ¹⁰Fn3 domain of a multivalent fibronectin based protein scaffoldbinds to a desired target with a K_(D) of less than 500 nM, 100 nM, 50nM, 1 nM, 500 μM, 100 μM or less. In some embodiments, each ¹⁰Fn3 domainof a multivalent fibronectin based protein scaffold binds to a desiredtarget with a K_(D) between 1 μM and 1 μM, between 100 μM and 500 nM,between 1 nM and 500 nM, or between 1 nM and 100 nM. In exemplaryembodiments, each ¹⁰Fn3 domain of a multivalent fibronectin basedprotein scaffold binds specifically to a target that is not bound by awild-type ¹⁰Fn3 domain, particularly the wild-type human ¹⁰Fn3 domain.

The ¹⁰Fn3 domains in a multivalent fibronectin based scaffold proteinmay be connected by a polypeptide linker. Exemplary polypeptide linkersinclude polypeptides having from 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3,or 1-2 amino acids. Suitable linkers for joining the ¹⁰Fn3 domains arethose which allow the separate domains to fold independently of eachother forming a three dimensional structure that permits high affinitybinding to a target molecule. Specific examples of suitable linkersinclude glycine-serine based linkers, glycine-proline based linkers,proline-alanine based linkers as well as linkers having the amino acidsequence PSTPPTPSPSTPPTPSPS (SEQ ID NO: 152). In some embodiments, thelinker is a glycine-serine based linker. In some embodiments, the linkeris a glycine-serine based linker. These linkers comprise glycine andserine residues and may be between 8 and 50, 10 and 30, and 10 and 20amino acids in length. Examples include linkers having an amino acidsequence (GS)₇ (SEQ ID NO: 153), G(GS)₆ (SEQ ID NO: 154), and G(GS)7G(SEQ ID NO: 155). Other linkers contain glutamic acid, and include, forexample, (GSE)₅ (SEQ ID NO: 156) and GGSEGGSE (SEQ ID NO: 157). Otherexemplary glycine-serine linkers include (GS)₄ (SEQ ID NO: 158),(GGGGS)₇ (SEQ ID NO: 159), (GGGGS)₅ (SEQ ID NO: 160), and (GGGGS)₃G (SEQID NO: 161). In some embodiments, the linker is a glycine-proline basedlinker. These linkers comprise glycine and proline residues and may bebetween 3 and 30, 10 and 30, and 3 and 20 amino acids in length.Examples include linkers having an amino acid sequence (GP)₃G (SEQ IDNO: 162), (GP)₅G (SEQ ID NO: 163), and GPG. In certain embodiments, thelinker may be a proline-alanine based linker having between 3 and 30, 10and 30, and 3 and 20 amino acids in length. Examples of proline alaninebased linkers include, for example, (PA)₃ (SEQ ID NO: 164), (PA)₆ (SEQID NO: 165) and (PA)₉ (SEQ ID NO: 166). It is contemplated, that theoptimal linker length and amino acid composition may be determined byroutine experimentation by methods well known in the art. In exemplaryembodiments, the linker does not contain any Asp-Lys (DK) pairs.

In certain embodiments, the linker has the amino acid sequencePSPEPPTPEP (SEQ ID NO: 173), PSPEPPTPEPPSPEPPTPEP (SEQ ID NO: 174),PSPEPPTPEPPSPEPPTPEPPSPEPPTPEP (SEQ ID NO: 175), orPSPEPPTPEPPSPEPPTPEPPSPEPPTPEPPSPEPPTPEP (SEQ ID NO: 176). Generally alinker may comprise the amino acid sequence (PSPEPPTPEP)_(o) (SEQ ID NO:262), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1-5 or 1-10. Incertain embodiments, the linker has the amino acid sequence EEEEDE (SEQID NO: 177), EEEEDEEEEDE (SEQ ID NO: 178), EEEEDEEEEDEEEEDEEEEDE (SEQ IDNO: 179), EEEEDEEEEDEEEEDEEEEDEEEEDEEEEDE (SEQ ID NO: 180). Generally, alinker may comprise the sequence (EEEEDE).E (SEQ ID NO: 263), wherein nis 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1-5 or 1-10. In certain embodiments,the linker has the amino acid sequence RGGEEKKKEKEKEEQEERETKTP (SEQ IDNO: 181). Such linkers may be used to connect the albumin bindingAdnectin to another polypeptide (e.g., another Adnectin). Exemplary usesof the PSPEPPTPEP (SEQ ID NO: 173) linker is shown below.

N-Terminal Adnectin Connected to C-Terminal Polypeptide:

(SEQ ID NO: 182) . . . NYRTPGPSPEPPTPEP-polypeptide

N-Terminal Polypeptide Connected to C-Terminal Adnectin:

(SEQ ID NO: 183) polypeptide-PSPEPPTPEPGVSDV . . .

In some embodiments, the multivalent Adnectin is a tandem Adnectincomprising a first ¹⁰Fn3 domain which binds to a serum albumin (e.g., aPKE2 Adnectin), and a second ¹⁰Fn3 domain that binds to a specifictarget. Tandem Adnectins may have the configuration albumin bindingAdnectin-X and X-albumin binding Adnectin, wherein X is a targetspecific ¹⁰Fn3 domain. The skilled artisan would be familiar withmethods for testing the functional activity and assessing thebiophysical properties of such such tandem Adnectin molecules.

In one aspect, the invention provides a fusion polypeptide comprising afirst fibronectin type III tenth (¹⁰Fn3) domain and a second ¹⁰Fn3domain, wherein the first ¹⁰Fn3 domain comprises a) AB, BC, CD, DE, EF,and FG loops, b) a CD loop with an altered amino acid sequence relativeto the sequence of the corresponding loop of the human ¹⁰Fn3 domain, andc) wherein the polypeptide binds to human serum albumin with a K_(D) ofless than 500 nM. A “first” domain and a second “domain” may be in theN- to C-terminal or C- to N-terminal orientation.

In some embodiments, e.g., of multivalent Adnectins, the first ¹⁰Fn3domain comprises an amino acid sequence at least 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs:23-100, 184-209 and 235-260 or differs therefrom in at most 1, 1-2, 1-5,1-10 or 1-20 amino acids, e.g., amino acid deletions, additions orsubstitutions (e.g., conservative amino acid substitutions).

In some embodiments, the first ¹⁰Fn3 domain comprises the amino acidsequence of any one of SEQ ID NOs: 23-100, 184-209 and 235-260.

In a preferred embodiment, the first ¹⁰Fn3 domain comprises the aminoacid sequence of SEQ ID NO: 29, 55, 81, 190 or 241. In another preferredembodiment, the first ¹⁰Fn3 domain comprises the amino acid sequence ofSEQ ID NO: 36, 62, 88, 197 or 248.

In some embodiments, the multivalent Adnectin comprises a second ¹⁰Fn3domain that is a ¹⁰Fn3 domain that specifically binds to a targetprotein other than serum albumin.

In a preferred embodiment, the second ¹⁰Fn3 domain specifically binds toPCSK9.

Accordingly, in one embodiment, the second ¹⁰Fn3 domain comprises anamino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to SEQ ID NO: 168 or 261 or differs therefromin at most 1, 1-2, 1-5, 1-10 or 1-20 amino acids, e.g., amino aciddeletions, additions or substitutions (e.g., conservative amino acidsubstitutions).

Additional suitable ¹⁰Fn3 domains that bind to PCSK9 are disclosed in,e.g., WO2011/130354, the contents of which are herein incorporated byreference.

In one embodiment, the second ¹⁰Fn3 domain has the amino acid sequenceset forth in SEQ ID NO: 168 or 261.

In certain embodiments, the invention provides a PCSK9-serum albuminbinding tandem Adnectin comprising the amino acid sequence set forth inSEQ ID NO: 168 or 261, as well as PCSK9-serum albumin tandem Adnectinswith amino acid sequences at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical thereto or differs therefrom in at most1, 1-2, 1-5, 1-10 or 1-20 amino acids, e.g., amino acid deletions,additions or substitutions (e.g., conservative amino acidsubstitutions), wherein the tandem Adnectin retains binding to PCSK9 andserum albumin.

In one embodiment, the invention provides nucleic acids encoding aPCSK9-serum albumin binding tandem Adnectin comprising the nucleic acidsequence set forth in SEQ ID NO: 172, as well as nucleic acid sequencesat least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical thereto, wherein the encoded PCSK9-serum albumin bindingtandem Adnectin retains binding to PCSK9 and serum albumin. In someembodiments, the nucleotide substitutions do not alter the resultingtranslated amino acid sequence (i.e., silent mutations).

In one aspect, the serum albumin binding-based tandem Adnectins (e.g.,PCSK9-PKE2 tandem Adnectin) described herein bind to human serum albuminwith a K_(D) of less than 3 μM, 2.5 μM, 2 μM, 1.5 μM, 1 μM, 500 nM, 100nM, 50 nM, 10 nM, 1 nM, 500 μM, 100 μM, 100 μM, 50 μM, or 10 μM. TheK_(D) may be, e.g., in the range of 0.1 nM to 50 nM, 0.1 nM to 100 nM,0.1 nM to 1 μM, 0.5 nM to 50 nM, 0.5 nM to 100 nM, 0.5 nM to 1 μM, 1 nMto 50 nM, 1 nM to 100 nM or 1 nM to 1 μM.

In certain embodiments, the serum albumin binding-based tandem Adnectins(e.g., PCSK9-PKE2 tandem Adnectin) described herein may also bind serumalbumin from one or more of cynomolgus monkey, rhesus monkey, rat, ormouse.

In certain embodiments, the serum albumin binding-based tandem Adnectins(e.g., PCSK9-PKE2 tandem Adnectin) described herein bind to rhesus serumalbumin (RhSA) or cynomolgous monkey serum albumin (CySA) with a K_(D)of less than 3 μM, 2.5 μM, 2 μM, 1.5 μM, 1 μM, 500 nM, 100 nM, 50 nM, 10nM, 1 nM, 500 μM or 100 μM. The K_(D) may be, e.g., in the range of 0.1nM to 50 nM, 0.1 nM to 100 nM, 0.1 nM to 1 μM, 0.5 nM to 50 nM, 0.5 nMto 100 nM, 0.5 nM to 1 μM, 1 nM to 50 nM, 1 nM to 100 nM or 1 nM to 1μM.

In certain embodiments, the serum albumin binding-based tandem Adnectins(e.g., PCSK9-PKE2 tandem Adnectin) described herein bind to rhesus serumalbumin (RhSA), cynomolgous monkey serum albumin (CySA), and mouse serumalbumin (MSA) with a K_(D) of less than 3 μM, 2.5 μM, 2 μM, 1.5 μM, 1μM, 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 μM or 100 μM. The K_(D) maybe, e.g., in the range of 0.1 nM to 50 nM, 0.1 nM to 100 nM, 0.1 nM to 1μM, 0.5 nM to 50 nM, 0.5 nM to 100 nM, 0.5 nM to 1 μM, 1 nM to 50 nM, 1nM to 100 nM or 1 nM to 1 μM.

In certain embodiments, the serum albumin binding-based tandem Adnectins(e.g., PCSK9-PKE2 tandem Adnectin) described herein bind to rhesus serumalbumin (RhSA), cynomolgous monkey serum albumin (CySA), mouse serumalbumin (MSA), and rat serum albumin (RSA) with a K_(D) of less than 3μM, 2.5 μM, 2 μM, 1.5 μM, 1 μM, 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500μM or 100 μM. The K_(D) may be, e.g., in the range of 0.1 nM to 50 nM,0.1 nM to 100 nM, 0.1 nM to 1 μM, 0.5 nM to 50 nM, 0.5 nM to 100 nM, 0.5nM to 1 μM, 1 nM to 50 nM, 1 nM to 100 nM or 1 nM to 1 μM.

In certain embodiments, the serum albumin binding-based tandem Adnectins(e.g., PCSK9-PKE2 tandem Adnectin) described herein bind to serumalbumin at a pH range of 5.5 to 7.4.

In certain embodiments, the tandem serum albumin binding-based Adnectins(e.g., PCSK9-PKE2 tandem Adnectin) described herein bind to domain I-IIof human serum albumin.

In certain embodiments, the tandem serum albumin binding-based Adnectins(e.g., PCSK9-PKE2 tandem Adnectin) described herein has a serumhalf-life in the presence of human serum albumin, cynomolgus monkeyserum albumin, rhesus monkey serum albumin, mouse serum albumin, and/orrat serum albumin of at least 1 hour, 2 hours, 5 hours, 10 hours, 20hours, 30 hours, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90hours, 100 hours, 150 hours, 200 hours, or at least about 300 hours. Incertain embodiments, the tandem serum albumin binding-based Adnectins(e.g., PCSK9-PKE2 tandem Adnectin) described herein has a serumhalf-life in the presence of human serum albumin, cynomolgus monkeyserum albumin, rhesus monkey serum albumin, mouse serum albumin, and/orrat serum albumin of 1-300 hours, such as 1-250 hours, 1-200 hours,1-150 hours, 1-100 hours, 1-90 hours, 1-80 hours, 1-70 hours, 1-60hours, 1-50 hours, 1-40 hours, 1-30 hours, 1-20 hours, 1-10 hours, 1-5hours, 5-300 hours, 10-300 hours, 20-300 hours, 30-300 hours, 40-300hours, 50-300 hours, 60-300 hours, 70-300 hours, 80-300 hours, 90-300hours, 100-300 hours, 150-300 hours, 200-300 hours, 250-300 hours, 5-250hours, 10-200 hours, 50-150 hours, or 80-120 hours.

In certain embodiments, the serum half-life of the partner Adnectin inthe serum albumin-based tandem Adnectin (e.g., PCSK9 Adnectin in thecase of a PCSK9-PKE2 tandem Adnectin) is increased relative to the serumhalf-life of the partner Adnectin when not conjugated to the serumalbumin binding Adnectin. In certain embodiments, the serum half-life ofthe serum albumin-based tandem Adnectin is at least 20, 40, 60, 80, 100,120, 150, 180, 200, 400, 600, 800, 1000, 1200, 1500, 1800, 1900, 2000,2500, or 3000% longer relative to the serum half-life of the partnerAdnectin when not fused to the serum albumin binding Adnectin. Incertain embodiments, the serum half-life of the serum albumin-basedtandem Adnectin is 20-3000%, such as 40-3000%, 60-3000%, 80-3000%,100-3000%, 120-3000%, 150-3000%, 180-3000%, 200-3000%, 400-3000%,600-3000%, 800-3000%, 1000-3000%, 1200-3000%, 1500-3000%, 1800-3000%,1900-3000%, 2000-3000%, 2500-3000%, 20-2500%, 20-2000%, 20-1900%,20-1800%, 20-1500%, 20-1200%, 20-1000%, 20-800%, 20-600%, 20-400%,20-200%, 20-180%, 20-150%, 20-120%, 20-100%, 20-80%, 20-60%, 20-40%,50-2500%, 100-2000%, 150-1500%, 200-1000%, 400-800%, or 500-700% longerrelative to the serum half-life of the partner Adnectin when not fusedto the serum albumin binding Adnectin. In certain embodiments, the serumhalf-life of the serum albumin binding-based tandem Adnectin is at least1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5 fold, 4-fold, 4.5-fold, 5-fold,6-fold, 7-fold, 8-fold, 10-fold, 12-fold, 13-fold, 15-fold, 17-fold,20-fold, 22-fold, 25-fold, 27-fold, 30-fold, 35-fold, 40-fold, or50-fold greater than the serum half-life of the partner Adnectin whennot fused to the serum albumin binding Adnectin. In certain embodiments,the serum half-life of the serum albumin binding-based tandem Adnectinis 1.5-50 fold, such as 1.5-40 fold, 1.5-35 fold, 1.5-30 fold, 1.5-27fold, 1.5-25 fold, 1.5-22 fold, 1.5-20 fold, 1.5-17 fold, 1.5-15 fold,1.5-13 fold, 1.5-12 fold, 1.5-10 fold, 1.5-9 fold, 1.5-8 fold, 1.5-7fold, 1.5-6 fold, 1.5-5 fold, 1.5-4.5 fold, 1.5-4 fold, 1.5-3.5 fold,1.5-3 fold, 1.5-2.5 fold, 1.5-2 fold, 2-50 fold, 2.5-50 fold, 3-50 fold,3.5-50 fold, 4-50 fold, 4.5-50 fold, 5-50 fold, 6-50 fold, 7-50 fold,8-50 fold, 10-50 fold, 12-50 fold, 13-50 fold, 15-50 fold, 17-50 fold,20-50 fold, 22-50 fold, 25-50 fold, 27-50 fold, 30-50 fold, 40-50 fold,2-40 fold, 5-35 fold, 10-20 fold, or 10-15 fold greater than the serumhalf-life of the partner Adnectin when not fused to the serum albuminbinding Adnectin. In certain embodiments, the serum half-life of theserum albumin binding-based tandem Adnectin is at least 2 hours, 2.5hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 50hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200hours. In certain embodiments, the serum half-life of the serum albuminbinding-based tandem Adnectin is 2-200 hours, 2.5-200 hours, 3-200hours, 4-200 hours, 5-200 hours, 6-200 hours, 7-200 hours, 8-200 hours,9-200 hours, 10-200 hours, 15-200 hours, 20-200 hours, 25-200 hours,30-200 hours, 35-200 hours, 40-200 hours, 50-200 hours, 60-200 hours,70-200 hours, 80-200 hours, 90-200 hours, 100-200 hours, 125-200 hours,150-200 hours, 175-200 hours, 190-200 hours, 2-190 hours, 2-175 hours,2-150 hours, 2-125 hours, 2-100 hours, 2-90 hours, 2-80 hours, 2-70hours, 2-60 hours, 2-50 hours, 2-40 hours, 2-35 hours, 2-30 hours, 2-25hours, 2-20 hours, 2-15 hours, 2-10 hours, 2-9 hours, 2-8 hours, 2-7hours, 2-6 hours, 2-5 hours, 2-4 hours, 2-3 hours, 5-175 hours, 10-150hours, 15-125 hours, 20-100 hours, 25-75 hours, or 30-60 hours.

E. Conjugates of Serum Albumin Binding Adnectins

Certain aspects of the present invention provide for conjugatescomprising a serum albumin binding Adnectin and at least one additionalmoiety (e.g., a therapeutic moiety). The additional moiety may be usefulfor any diagnostic, imaging, or therapeutic purpose.

In some embodiments, the serum albumin binding Adnectin is fused to asecond moiety that is a small organic molecule, a nucleic acid, apeptide, or a protein. In some embodiments, the serum albumin bindingAdnectin is fused to a therapeutic moiety that targets receptors,receptor ligands, viral coat proteins, immune system proteins, hormones,enzymes, antigens, or cell signaling proteins. The fusion may be formedby attaching the second moiety to either end of the serum albuminbinding Adnectin, i.e., serum albumin binding Adnectin-therapeuticmolecule or therapeutic molecule-serum albumin binding Adnectinarrangements.

In certain embodiments, the serum half-life of the moiety fused to theserum albumin binding Adnectin is increased relative to the serumhalf-life of the moiety when not conjugated to the serum albumin bindingAdnectin. In certain embodiments, the serum half-life of the serumalbumin binding Adnectin fusion is at least 20, 40, 60, 80, 100, 120,150, 180, 200, 400, 600, 800, 1000, 1200, 1500, 1800, 1900, 2000, 2500,or 3000% longer relative to the serum half-life of the moiety when notfused to the serum albumin binding Adnectin. In certain embodiments, theserum half-life of the serum albumin binding Adnectin fusion is20-3000%, such as 40-3000%, 60-3000%, 80-3000%, 100-3000%, 120-3000%,150-3000%, 180-3000%, 200-3000%, 400-3000%, 600-3000%, 800-3000%,1000-3000%, 1200-3000%, 1500-3000%, 1800-3000%, 1900-3000%, 2000-3000%,2500-3000%, 20-2500%, 20-2000%, 20-1900%, 20-1800%, 20-1500%, 20-1200%,20-1000%, 20-800%, 20-600%, 20-400%, 20-200%, 20-180%, 20-150%, 20-120%,20-100%, 20-80%, 20-60%, 20-40%, 50-2500%, 100-2000%, 150-1500%,200-1000%, 400-800%, or 500-700% longer relative to the serum half-lifeof the moiety when not fused to the serum albumin binding Adnectin. Incertain embodiments, the serum half-life of the PKE2 Adnectin fusion isat least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5 fold, 4-fold, 4.5-fold,5-fold, 6-fold, 7-fold, 8-fold, 10-fold, 12-fold, 13-fold, 15-fold,17-fold, 20-fold, 22-fold, 25-fold, 27-fold, 30-fold, 35-fold, 40-fold,or 50-fold greater than the serum half-life of the moiety when not fusedto the serum albumin binding Adnectin. In certain embodiments, the serumhalf-life of the PKE2 Adnectin fusion is 1.5-50 fold, such as 1.5-40fold, 1.5-35 fold, 1.5-30 fold, 1.5-27 fold, 1.5-25 fold, 1.5-22 fold,1.5-20 fold, 1.5-17 fold, 1.5-15 fold, 1.5-13 fold, 1.5-12 fold, 1.5-10fold, 1.5-9 fold, 1.5-8 fold, 1.5-7 fold, 1.5-6 fold, 1.5-5 fold,1.5-4.5 fold, 1.5-4 fold, 1.5-3.5 fold, 1.5-3 fold, 1.5-2.5 fold, 1.5-2fold, 2-50 fold, 2.5-50 fold, 3-50 fold, 3.5-50 fold, 4-50 fold, 4.5-50fold, 5-50 fold, 6-50 fold, 7-50 fold, 8-50 fold, 10-50 fold, 12-50fold, 13-50 fold, 15-50 fold, 17-50 fold, 20-50 fold, 22-50 fold, 25-50fold, 27-50 fold, 30-50 fold, 40-50 fold, 2-40 fold, 5-35 fold, 10-20fold, or 10-15 fold greater than the serum half-life of the moiety whennot fused to the serum albumin binding Adnectin. In some embodiments,the serum half-life of the serum albumin binding Adnectin fusion is atleast 2 hours, 2.5 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8hours, 9 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35hours, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100hours, 110 hours, 120 hours, 130 hours, 135 hours, 140 hours, 150 hours,160 hours, or 200 hours. In certain embodiments, the serum half-life ofthe serum albumin binding Adnectin fusion is 2-200 hours, 2.5-200 hours,3-200 hours, 4-200 hours, 5-200 hours, 6-200 hours, 7-200 hours, 8-200hours, 9-200 hours, 10-200 hours, 15-200 hours, 20-200 hours, 25-200hours, 30-200 hours, 35-200 hours, 40-200 hours, 50-200 hours, 60-200hours, 70-200 hours, 80-200 hours, 90-200 hours, 100-200 hours, 125-200hours, 150-200 hours, 175-200 hours, 190-200 hours, 2-190 hours, 2-175hours, 2-150 hours, 2-125 hours, 2-100 hours, 2-90 hours, 2-80 hours,2-70 hours, 2-60 hours, 2-50 hours, 2-40 hours, 2-35 hours, 2-30 hours,2-25 hours, 2-20 hours, 2-15 hours, 2-10 hours, 2-9 hours, 2-8 hours,2-7 hours, 2-6 hours, 2-5 hours, 2-4 hours, 2-3 hours, 5-175 hours,10-150 hours, 15-125 hours, 20-100 hours, 25-75 hours, or 30-60 hours.

In certain embodiments, the serum albumin binding Adnectin fusionproteins bind to HSA with a K_(D) of less than 3 μM, 2.5 μM, 2 μM, 1.5μM, 1 μM, 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 μM, 100 μM, 100 μM, 50μM or 10 μM. The K_(D) may be, e.g., in the range of 0.1 nM to 50 nM,0.1 nM to 100 nM, 0.1 nM to 1 μM, 0.5 nM to 50 nM, 0.5 nM to 100 nM, 0.5nM to 1 μM, 1 nM to 50 nM, 1 nM to 100 nM or 1 nM to 1 μM.

In some embodiments, a therapeutic moiety may be directly or indirectlylinked to a serum albumin binding Adnectin via a polymeric linker, asdescribed herein. Polymeric linkers can be used to optimally vary thedistance between each component of the fusion to create a protein fusionwith one or more of the following characteristics: 1) reduced orincreased steric hindrance of binding of one or more protein domainswhen binding to a protein of interest, 2) increased protein stability orsolubility, 3) decreased protein aggregation, and 4) increased overallavidity or affinity of the protein.

In some embodiments, the fusions described herein are linked to theserum albumin binding Adnectin via a polypeptide linker having aprotease site that is cleavable by a protease in the blood or targettissue. Such embodiments can be used to release a therapeutic proteinfor better delivery or therapeutic properties or more efficientproduction.

Additional linkers or spacers may be introduced at the C-terminus of an¹⁰Fn3 domain between the ¹⁰Fn3 domain and the polypeptide linker.

In some embodiments, a therapeutic moiety is linked to a serum albuminbinding Adnectin via a biocompatible polymer such as a polymeric sugar.The polymeric sugar can include an enzymatic cleavage site that iscleavable by an enzyme in the blood or target tissue. Such embodimentscan be used to release therapeutic proteins for better delivery ortherapeutic properties or more efficient production.

The serum albumin binding Adnectin fusion molecules described herein areuseful for increasing the half-life of a therapeutic moiety by creatinga fusion between the therapeutic moiety and the serum albumin bindingAdnectin. Such fusion molecules may be used to treat conditions whichrespond to the biological activity of the therapeutic moiety containedin the fusion. The present invention contemplates the use of the serumalbumin binding Fn3 fusion molecules in diseases caused by thedisregulation of any of the following proteins or molecules.

In exemplary embodiments, the therapeutic moiety that is linked (eitherC-terminal or N-terminal) to the serum albumin binding Adnectin is VEGF,VEGF-R1, VEGF-R2, VEGF-R3, Her-1, Her-2, Her-3, EGF-I, EGF-2, EGF-3,Alpha3, cMet, ICOS, CD40L, LFA-I, c-Met, ICOS, LFA-I, IL-6, B7.1, W1.2,OX40, IL-1b, TACI, IgE, BAFF or BLys, TPO-R, CD19, CD20, CD22, CD33,CD28, IL-I-R1, TNF-alpha, TRAIL-R1, Complement Receptor 1, FGFa,Osteopontin, Vitronectin, Ephrin A1-A5, Ephrin B1-B3,alpha-2-macroglobulin, CCL, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CXCL,CXCL9, CXCL10, CXCL11, CXCL2, CCL13, CCL4, CCL5, CXCL16, CCL16, CCL17,CCL18, CCL19, CCL20, CCL21, CCL22, PDGF, TGFb, GMCSF, SCF, p40(IL12/IL23), IL1b, IL1a, IL1ra, IL2, IL3, IL4, IL5, IL6, IL, IL, 10,IL12, IL15, IL23, Fas, FasL, Flt3 ligand, 41BB, ACE, ACE-2, KGF, FGF-7,SCF, Netrin1,2, IFNa,b,g, Caspase-2,3,7,8,10, ADAM S1,S5,8,9,15,TS1,TS5;Adiponectin, ALCAM, ALK-I, APRIL, Annexin V, Angiogenin, Amphiregulin,Angiopoietin-1,2,4, B7-1/CD80, B7-2/CD86, B7-H1, B7-H2, B7-H3, Bcl-2,BACE-I, BAK, BCAM, BDNF, bNGF, bECGF, BMP2,3,4,5,6,7,8; CRP, Cadherin 6,8, 11; Cathepsin A,B,C,D,E,L,S,V,X; CD 1la/LFA-1, LFA-3, GP2b3a, GHreceptor, RSV F protein, IL-23 (p40, p19), IL-12, CD80, CD86, CD28,CTLA-4, alpha4-beta1, alpha4-beta7, TNF/Lymphotoxin, IgE, CD3, CD20,IL-6, IL-6R, BLYS/BAFF, IL-2R, HER2, EGFR, CD33, CD52, Digoxin, Rho (D),Varicella, Hepatitis, CMV, Tetanus, Vaccinia, Antivenom, Botulinum,Trail-R1, Trail-R2, cMet, TNF-R family, such as LA NGF-R, CD27, CD30,CD40, CD95, Lymphotoxin a/b receptor, WsI-I, TLIAITNFSF15, BAFF,BAFF-R/TNFRSF13C, TRAIL R2ITNFRSF10B, TRAIL R2ITNFRSF10B, FasITNFRSF6CD27/TNFRSF7, DR3/TNFRSF25, HVEMTNFRSF14, TROY/TNFRSF19, CD40Ligand/TNFSF5, BCMA/TNFRSF17, CD30/TNFRSF8, LIGHT/TNFSF14,4-1BB/TNFRSF9, CD40/TNFRSF5, GITR/[Gamma]NFRSF 18,Osteoprotegerin/TNFRSF1 IB, RANKTNFRSF1 IA, TRAIL R3/TNFRSFIOC,TRAIIJTNFSFIO, TRANCE/RANK LITNFSF11, 4-1BB Ligand/TNFSF9,TWEAK/TNFSF12, CD40 LigandlTNFSFS, Fas LigandITNFSF6, RELT/TNFRSF19L,APRIUITNFSF13, DcR3/TNFRSF6B, TNF R1/TNFRSFIA, TRAIL R1/TNFRSFIOA, TRAILR4/TNFRSF10D, CD30 LigandITNFSF8, GITR LigandITNFSF18, TNFSF18,TACI/TNFRSF13B, NGF RTNFRSF16, OX40 Ligand/TNFSF4, TRAIL R2ITNFRSF10B,TRAIL R3/TNFRSF10C, TWEAK RITNFRSF12, BAFF/BLyS/TNFSF13, DR6/TNFRSF21,TNF-alpha/TNFSF1 A, Pro-TNF-alpha/TNFSF1A, Lymphotoxin beta RTNFRSF3,Lymphotoxin beta R (LTbR)/Fc Chimera, TNF RITNFRSFIA, TNF-beta/TNFSF1B,PGRP-S, TNF RI/TNFRSFIA, TNF RII/TNFRSFIB, EDA-A2, TNF-alpha/TNFSFIA,EDAR, XEDAR, TNF RI/TNFRSFIA.

In exemplary embodiments, the therapeutic moiety that is linked (eitherC-terminal or N-terminal) to the serum albumin binding Adnectin is anyof the following proteins or proteins binding thereto: 4EBP1, 14-3-3zeta, 53BP1, 2B4/SLAMF4, CCL21/6Ckine, 4-1BB/TNFRSF9, 8D6A, 4-1BBLigandITNFSF9, 8-oxo-dG, 4-Amino-1,8-naphthalimide, A2B5, AminopeptidaseLRAP/ERAP2, A33, Aminopeptidase N/ANPEP, Aag, Aminopeptidase P2/XPNPEP2,ABCG2, Aminopeptidase P1/XPNPEP1, ACE, Aminopeptidase PILS/ARTS1, ACE-2,Amnionless, Actin, Amphiregulin, beta-Actin, AMPK alpha 1/2, Activin A,AMPK alpha 1, Activin AB, AMPK alpha 2, Activin B, AMPK beta 1, ActivinC, AMPK beta 2, Activin RIA/ALK-2, Androgen R/NR3C4, Activin RIB/ALK-4,Angiogenin, Activin RIA, Angiopoietin-1, Activin RIIB, Angiopoietin-2,ADAMS, Angiopoietin-3, ADAM9, Angiopoietin-4, ADAM 10, Angiopoietin-like1, ADAM 12, Angiopoietin-like 2, ADAM 15, Angiopoietin-like 3, TACE/ADAM17, Angiopoietin-like 4, ADAM 19, Angiopoietin-like 7/CDT6, ADAM33,Angiostatin, ADAMTS4, Annexin A1/Annexin I, ADAMTS5, Annexin A7,ADAMTS1, Annexin A10, ADAMTSL-1/Punctin, Annexin V, Adiponectin/Acrp30,ANP, AEBSF, AP Site, Aggrecan, APAF-I, Agrin, APC, AgRP, APE, AGTR-2,APJ, AIF, APLP-I, Akt, APLP-2, Akt1, Apolipoprotein AL, Akt2,Apolipoprotein B, Akt3, APP, Serum Albumin, APRIUITNFSF13, ALCAM, ARC,ALK-I, Artemin, ALK-7, Arylsulfatase AJARSA, Alkaline Phosphatase,ASAH2/N-acylsphingosine Amidohydrolase-2, alpha 2u-Globulin, ASC,alpha-i-Acid Glycoprotein, ASGR1, alpha-Fetoprotein, ASKi, ALS, ATM,Ameloblastic ATRIP, AMICA/JAML, Aurora A, AMIGO, Aurora B, AMIG02,Axin-1, AMIG03, AxI, Aminoacylase/ACY1, Azurocidin/CAP37/HBP,Aminopeptidase A/ENPEP, B4GALT, BIM, B7-1/CD80, 6-Biotin-17-NAD,B7-2/CD86, BLAME/SLAMF8, B7-H1/PD-L1, CXCL13/BLC/BCA-1, B7-H2, BLIMPI,B7-H3, BIk, B7-H4, BMI-I, BACE-I, BMP-1/PCP, BACE-2, BMP-2, Bad, BMP-3,BAFF/TNFSF13B, BMP-3b/GDF-10, BAFF R/TNFRSF 13C, BMP-4, Bag-1, BMP-5,BAK, BMP-6, BAMBI/NMA, BMP-7, BARD 1, BMP-8, Bax, BMP-9, BCAM, BMP-10,Bcl-10, BMP-15/GDF-9B, Bcl-2, BMPR-IA/ALK-3, Bcl-2 related protein A1,BMPR-IB/ALK-6, Bcl-w, BMPR-II, Bcl-x, BNIP3L, Bcl-xL, BOC,BCMA/TNFRSF17, BOK, BDNF, BPDE, Benzamide, Brachyury, Common beta Chain,B-Raf, beta IG-H3, CXCL14/BRAK, Betacellulin, BRCA1, beta-Defensin 2,BRCA2, BID, BTLA, Biglycan, Bub-1, Bik-like Killer Protein, c-jun,CD90Thyl, c-Rel, CD94, CCL6/C10, CD97, CIq R1/CD93, CD151, CIqTNF,CD160, ClqTNF4, CD163, ClqTNF5, CD 164, Complement Component CIr, CD200,Complement Component CIs, CD200 R1, Complement Component C2,CD229/SLAMF3, Complement Component C3a, CD23/Fc epsilon R11, ComplementComponent C3d, CD2F-10/SLAMF9, Complement Component C5a, CD5L,Cadherin-4/R-Cadherin, CD69, Cadherin-6, CDC2, Cadherin-8, CDC25A,Cadherin-I 1, CDC25B, Cadherin-12, CDCP1, Cadherin-13, CDO, Cadherin-17,CDX4, E-Cadherin, CEACAM-1/CD66a, N-Cadherin, CEACAM-6, P-Cadherin,Cerberus 1, VE-Cadherin, CFTR, Calbindin D, cGMP, Calcineurin A, ChemR23, Calcineurin B, Chemerin, Calreticulin-2, Chemokine Sampler Packs,CaM Kinase II, Chitinase 3-like 1, cAMP, Chitotriosidase/CHIT1,Cannabinoid R1, Chk1, Cannabinoid R2/CB2/CNR2, Chk2, CAR/NR1I3,CHL-1/L1CAM-2, Carbonic Anhydrase I, Choline Acetyltransferase/CbAT,Carbonic Anhydrase II, Chondrolectin, Carbonic Anhydrase III, Chordin,Carbonic Anhydrase IV, Chordin-Like 1, Carbonic Anhydrase VA,Chordin-Like 2, Carbonic Anhydrase VB, CINC-I, Carbonic Anhydrase VI,CINC-2, Carbonic Anhydrase VII, CINC-3, Carbonic Anhydrase VIII,Claspin, Carbonic Anhydrase IX, Claudin-6, Carbonic Anhydrase X, CLC,Carbonic Anhydrase XII, CLEC-I, Carbonic Anhydrase XIII, CLEC-2,Carbonic Anhydrase XIV, CLECSF 13/CLEC4F, Carboxymethyl Lysine, CLECSF8,Carboxypeptidase A1/CPA1, CLF-I, Carboxypeptidase A2, CL-P1/COLEC12,Carboxypeptidase A4, Clusterin, Carboxypeptidase B1, Clusterin-like 1,Carboxypeptidase E/CPE, CMG-2, Carboxypeptidase X1, CMV UL146,Cardiotrophin-1, CMV UL147, Carnosine Dipeptidase 1, CNP, Caronte, CNTF,CART, CNTF R alpha, Caspase, Coagulation Factor IIThrombin, Caspase-1,Coagulation Factor I11/Tissue Factor, Caspase-2, Coagulation Factor VII,Caspase-3, Coagulation Factor X, Caspase-4, Coagulation Factor XI,Caspase-6, Coagulation Factor XIV/Protein C, Caspase-7, COCO, Caspase-8,Cohesin, Caspase-9, Collagen I, Caspase-10, Collagen II, Caspase-12,Collagen IV, Caspase-13, Common gamma Chain/IL-2 R gamma, CaspasePeptide Inhibitors, COMP/Thrombospondin-5, Catalase, ComplementComponent CIrLP, beta-Catenin, Complement Component CIqA, Cathepsin 1,Complement Component CIqC, Cathepsin 3, Complement Factor D, Cathepsin6, Complement Factor I, Cathepsin A, Complement MASP3, Cathepsin B,Connexin 43, Cathepsin C/DPPI, Contactin-1, Cathepsin D, Contactin-2/TAG1, Cathepsin E, Contactin-4, Cathepsin F, Contactin-5, Cathepsin H,Corin, Cathepsin L, Cornulin, Cathepsin O, CORS26/ClqTNF,3, Cathepsin S,Rat Cortical Stem Cells, Cathepsin V, Cortisol, Cathepsin XITJ?, COUP-TFI/NR2F1, CBP, COUP-TF II/NR2F2, CCI, COX-I, CCK-A R, COX-2, CCL28,CRACC/SLAMF7, CCR1, C-Reactive Protein, CCR2, Creatine Kinase,Muscle/CKMM, CCR3, Creatinine, CCR4, CREB, CCR5, CREG, CCR6, CRELD,CCR7, CRELD2, CCR8, CRHBP, CCR9, CRHR-I, CCR1O, CRIMI, CD155/PVR,Cripto, CD2, CRISP-2, CD3, CRISP-3, CD4, Crossveinless-2, CD4+/45RA-,CRTAM, CD4+/45RO, CRTH-2, CD4+/CD62L-/CD44, CRY1, CD4+/CD62L+/CD44,Cryptic, CD5, CSB/ERCC6, CD6, CCL27/CTACK, CD8, CTGF/CCN2, CD8+/45RA-,CTLA-4, CD8+/45RO-, Cubilin, CD9, CX3CR1, CD14, CXADR, CD27/TNFRSF7,CXCL16, CD27 Ligand/TNFSF7, CXCR3, CD28, CXCR4, CD30/TNFRSF8, CXCR5,CD30 Ligand/TNFSF8, CXCR6, CD31/PECAM-1, Cyclophilin A, CD34,Cyr61/CCN1, CD36/SR-B3, Cystatin A, CD38, Cystatin B, CD40/TNFRSF5,Cystatin C, CD40 Ligand/TNFSF5, Cystatin D, CD43, Cystatin E/M, CD44,Cystatin F, CD45, Cystatin H, CD46, Cystatin H2, CD47, Cystatin S,CD48/SLAMF2, Cystatin SA, CD55/DAF, Cystatin SN, CD58/LFA-3, Cytochromec, CD59, Apocytochrome c, CD68, Holocytochrome c, CD72, Cytokeratin 8,CD74, Cytokeratin 14, CD83, Cytokeratin 19, CD84/SLAMF5, Cytonin, D6,DISPI, DAN, Dkk-1, DANCE, Dkk-2, DARPP-32, Dkk-3, DAX1/NR0B1, Dkk-4,DCC, DLEC, DCIR/CLEC4A, DLL1, DCAR, DLL4, DcR3/TNFRSF6B, d-Luciferin,DC-SIGN, DNA Ligase IV, DC-SIGNR/CD299, DNA Polymerase beta, DcTRAILR1ITNFRSF23, DNAM-I, DcTRAIL R2/TNFRSF22, DNA-PKcs, DDR1, DNER, DDR2,Dopa Decarboxylase/DDC, DEC-205, DPCR-I, Decapentaplegic, DPP6, Decorin,DPP A4, Dectin-1/CLEC7A, DPPA5/ESGI, Dectin-2/CLEC6A, DPPII/QPP/DPP7,DEP-1/CD148, DPPIV/CD26, Desert Hedgehog, DR3/TNFRSF25, Desmin,DR6/TNFRSF21, Desmoglein-1, DSCAM, Desmoglein-2, DSCAM-L1, Desmoglein-3,DSPG3, Dishevelled-1, Dtk, Dishevelled-3, Dynamin, EAR2/NR2F6, EphA5,ECE-I, EphA6, ECE-2, EphA7, ECF-UCHI3L3, EphA8, ECM-I, EphB1, Ecotin,EphB2, EDA, EphB3, EDA-A2, EphB4, EDAR, EphB6, EDG-I, Ephrin, EDG-5,Ephrin-A1, EDG-8, Ephrin-A2, eEF-2, Ephrin-A3, EGF, Ephrin-A4, EGF R,Ephrin-A5, EGR1, Ephrin-B, EG-VEGF/PK1, Ephrin-B 1, eIF2 alpha,Ephrin-B2, eIF4E, Ephrin-B3, Elk-I, Epigen, EMAP-II, Epimorphin/Syntaxin2, EMMPRIN/CD147, Epiregulin, CXCL5/ENA, EPR-I/Xa Receptor, Endocan,ErbB2, Endoglin/CD 105, ErbB3, Endoglycan, ErbB4, Endonuclease III, ERCC1, Endonuclease IV, ERCC3, Endonuclease V, ERK1/ERK2, Endonuclease VIII,ERK1, Endorepellin/Perlecan, ERK2, Endostatin, ERK3, Endothelin-1,ERK5/BMKI, Engrailed-2, ERR alpha/NR3B1, EN-RAGE, ERR beta/NR3B2,Enteropeptidase/Enterokinase, ERR gamma/NR3B3, CCL1 1/Eotaxin,Erythropoietin, CCL24/Eotaxin-2, Erythropoietin R, CCL26/Eotaxin-3,ESAM, EpCAM/TROP-1, ER alpha/NR3A1, EPCR, ER beta/NR3A2, Eph,Exonuclease III, EphA1, Exostosin-like 2/EXTL2, EphA2, Exostosin-like3/EXTL3, EphA3, FABP1, FGF-BP, FABP2, FGF R1-4, FABP3, FGF R1, FABP4,FGF R2, FABP5, FGF R3, FABP7, FGF R4, FABP9, FGF R5, Complement FactorB, Fgr, FADD, FHR5, FAM3A, Fibronectin, FAM3B, Ficolin-2, FAM3C,Ficolin-3, FAM3D, FITC, Fibroblast Activation Protein alpha/FAP, FKBP38,Fas/TNFRSF6, Flap, Fas Ligand/TNFSF6, FLIP, FATP1, FLRG, FATP4, FLRT1,FATP5, FLRT2, Fc gamma R1/CD64, FLRT3, Fc gamma RIIB/CD32b, Flt-3, Fcgamma RIIC/CD32c, Fit-3 Ligand, Fc gamma RIIA/CD32a, Follistatin, Fcgamma RIII/CD16, Follistatin-like 1, FcRH1/IRTA5, FosB/GOS3,FcRH2/IRTA4, FoxD3, FcRH4/IRTA1, FoxJ1, FcRH5/IRTA2, FoxP3, FcReceptor-like 3/CD 16-2, Fpg, FEN-I, FPR1, Fetuin A, FPRL1, Fetuin B,FPRL2, FGF acidic, CX3CL1/Fractalkine, FGF basic, Frizzled-1, FGF-3,Frizzled-2, FGF-4, Frizzled-3, FGF-5, Frizzled-4, FGF-6, Frizzled-5,FGF-8, Frizzled-6, FGF-9, Frizzled-7, FGF-IO, Frizzled-8, FGF-11,Frizzled-9, FGF-12, Frk, FGF-13, sFRP-1, FGF-16, sFRP-2, FGF-17, sFRP-3,FGF-19, sFRP-4, FGF-20, Furin, FGF-21, FXR/NR1H4, FGF-22, Fyn, FGF-23,G9a/EHMT2, GFR alpha-3/GDNF R alpha-3, GABA-A-R alpha 1, GFRalpha-4/GDNF R alpha-4, GABA-A-R alpha 2, GITR/TNFRSF18, GABA-A-R alpha4, GITR Ligand/TNFSF18, GABA-A-R alpha 5, GLI-I, GABA-A-R alpha 6,GLI-2, GABA-A-R beta 1, GLP/EHMT1, GABA-A-R beta 2, GLP-I R, GABA-A-Rbeta 3, Glucagon, GABA-A-R gamma 2, Glucosamine(N-acetyl)-6-Sulfatase/GNS, GABA-B-R2, GIuR1, GAD1/GAD67, GluR2/3,GAD2/GAD65, GluR2, GADD45 alpha, GluR3, GADD45 beta, Glut1, GADD45gamma, Glut2, Galectin-1, Glut3, Galectin-2, Glut4, Galectin-3, Glut5,Galectin-3 BP, Glutaredoxin 1, Galectin-4, Glycine R, Galectin-7,Glycophorin A, Galectin-8, Glypican 2, Galectin-9, Glypican 3,GalNAc4S-6ST, Glypican 5, GAP-43, Glypican 6, GAPDH, GM-CSF, Gas1,GM-CSF R alpha, Gas6, GMF-beta, GASP-1/WFIKKNRP, gpl30, GASP-2/WFIKKN,Glycogen Phosphorylase BB/GPBB, GATA-I, GPR15, GATA-2, GPR39, GATA-3,GPVI, GATA-4, GR/NR3C1, GATA-5, Gr-1/Ly-6G, GATA-6, Granulysin, GBL,Granzyme A, GCNF/NR6A1, Granzyme B, CXCL6/GCP-2, Granzyme D, G-CSF,Granzyme G, G-CSF R, Granzyme H, GDF-I, GRASP, GDF-3 GRB2, GDF-5,Gremlin, GDF-6, GRO, GDF-7, CXCL1/GRO alpha, GDF-8, CXCL2/GRO beta,GDF-9, CXCL3/GRO gamma, GDF-11, Growth Hormone, GDF-15, Growth HormoneR, GDNF, GRP75/HSPA9B, GFAP, GSK-3 alpha/beta, GFI-I, GSK-3 alpha, GFRalpha-1/GDNF R alpha-1, GSK-3 beta, GFR alpha-2/GDNF R alpha-2, EZFIT,H2AX, Histidine, H60, HM74A, HAI-I, HMGA2, HAI-2, HMGB1, HAI-2A,TCF-2/HNF-1 beta, HAI-2B, HNF-3 beta/FoxA2, HAND1, HNF-4 alpha/NR2A1,HAPLN1, HNF-4 gamma/NR2A2, Airway Trypsin-like Protease/HAT,HO-1/HMOX1/HSP32, HB-EGF, HO-2/HMOX2, CCL 14a/HCC-1, HPRG, CCL14b/HCC-3,Hrk, CCL16/HCC-4, HRP-I, alpha HCG, HS6ST2, Hck, HSD-I, HCR/CRAM-A/B,HSD-2, HDGF, HSP 10/EPF, Hemoglobin, HSP27, Hepassocin, HSP60, HES-1,HSP70, HES-4, HSP90, HGF, HTRA/Protease Do, HGF Activator, HTRA1/PRSS11,HGF R, HTRA2/0 ml, HIF-I alpha, HVEM/TNFRSF14, HIF-2 alpha, Hyaluronan,HIN-1/Secretoglobulin 3A1, 4-Hydroxynonenal, Hip, CCL1/I-309/TCA-3,IL-IO, cIAP (pan), IL-IO R alpha, cIAP-1/HIAP-2, IL-10 R beta,cIAP-2/HIAP-1, IL-1, IBSP/Sialoprotein II, EL-11 R alpha, ICAM-1/CD54,IL-12, ICAM-2/CD102, IL-12/IL-23 p40, ICAM-3/CD50, IL-12 R beta 1,ICAM-5, IL-12 R beta 2, ICAT, IL-13, ICOS, IL-13 R alpha 1, Iduronate2-Sulfatase/EOS, IL-13 R alpha 2, EFN, IL-15, IFN-alpha, IL-15 R alpha,IFN-alpha 1, IL-16, IFN-alpha 2, IL-17, IFN-alpha 4b, IL-17 R, IFN-alphaA, IL-17 RC, IFN-alpha B2, IL-17 RD, IFN-alpha C, IL-17B, IFN-alpha D,IL-17B R, IFN-alpha F, IL-17C, IFN-alpha G, IL-17D, IFN-alpha H2,IL-17E, IFN-alpha I, IL-17F, IFN-alpha J 1, IL-18/IL-1F4, IFN-alpha K,IL-18 BPa, IFN-alpha WA, IL-18 BPc, IFN-alpha/beta R1, IL-18 BPd,IFN-alpha/beta R2, IL-18 R alpha/IL-1 R5, IFN-beta, IL-18 R beta/IL-1R7, IFN-gamma, IL-19, IFN-gamma R1, IL-20, IFN-gamma R2, IL-20 R alpha,IFN-omega, IL-20 R beta, IgE, IL-21, IGFBP-I, IL-21 R, IGFBP-2, IL-22,IGFBP-3, IL-22 R, IGFBP-4, IL-22BP, IGFBP-5, IL-23, IGFBP-6, IL-23 R,IGFBP-L1, IL-24, IGFBP-rp1/IGFBP-7, IL-26/AK155, IGFBP-rPIO, IL-27,IGF-I, EL-28A, IGF-I R, IL-28B, IGF-II, IL-29/EFN-lambda 1, IGF-II R,IL-3 1, IgG, EL-31 RA, IgM, IL-32 alpha, IGSF2, IL-33, IGSF4A/SynCAM,ILT2/CD85J, IGSF4B, ILT3/CD85k, IGSF8, ILT4/CD85d, IgY, ILT5/CD85a,IkB-beta, ILT6/CD85e, IKK alpha, Indian Hedgehog, IKK epsilon, INSRR,EKK gamma, Insulin, IL-1 alpha/IL-IF1, Insulin R/CD220, IL-1beta/IL-1F2, Proinsulin, IL-ira/IL-1F3, Insulysin/EDE, IL-F5/FIL1 delta,Integrin alpha 2/CD49b, IL-F6/FIL1 epsilon, Integrin alpha 3/CD49c,IL-1F7/FIL1 zeta, Integrin alpha 3 beta 1/VNLA-3, IL-1F8/FIL1 eta,Integrin alpha 4/CD49d, IL-1F9/IL-1 H1, Integrin alpha 5/CD49e,IL-1F10/IL-1HY2, Integrin alpha 5 beta 1, IL-I RI, Integrin alpha6/CD49f, IL-I RII, Integrin alpha 7, IL-I R3/IL-1 R AcP, Integrin alpha9, IL-I R4/ST2, Integrin alpha E/CD103, IL-I R6/IL-1 R rp2, Integrinalpha L/CD1 Ia, IL-I R8, Integrin alpha L beta 2, IL-I R9, Integrinalpha M/CD1 Ib, IL-2, Integrin alpha M beta 2, IL-2 R alpha, Integrinalpha V/CD5 1, IL-2 R beta, Integrin alpha V beta 5, IL-3, Integrinalpha V beta 3, IL-3 R alpha, Integrin alpha V beta 6, IL-3 R beta,Integrin alpha XJCD1 Ic, IL-4, Integrin beta 1/CD29, IL-4 R, Integrinbeta 2/CD18, IL-5, Integrin beta 3/CD61, IL-5 R alpha, Integrin beta 5,IL-6, Integrin beta 6, IL-6 R, Integrin beta 7, IL-7,CXCL10/EP-10/CRG-2, IL-7 R alpha/CD127, IRAKI, CXCR1/IL-8 RA, IRAK4,CXCR2/IL-8 RB, ERS-I, CXCL8/IL-8, Islet-1, IL-9, CXCL1 1/I-TAC, IL-9 R,Jagged 1, JAM-4/IGSF5, Jagged 2, JNK, JAM-A, JNK1/JNK2, JAM-BNE-JAM,JNK1, JAM-C, JNK2, Kininogen, Kallikrein 3/PSA, Kininostatin, Kallikrein4, KER/CD158, Kallikrein 5, KER2D1, Kallikrein 6/Neurosin, KIR2DL3,Kallikrein 7, KIR2DL4/CD158d, Kallikrein 8/Neuropsin, KIR2DS4,Kallikrein 9, KIR3DL1, Plasma Kallikrein/KLKB1, KER3DL2, Kallikrein 10,Kirrel2, Kallikrein 11, KLF4, Kallikrein 12, KLF5, Kallikrein 13, KLF6,Kallikrein 14, Klotho, Kallikrein 15, Klotho beta, KC, KOR, Keap1,Kremen-1, KeI1, Kremen-2, KGF/FGF-7, LAG-3, LINGO-2, LAIR, Lipin 2,LAIR2, Lipocalin-1, Laminin alpha 4, Lipocalin-2/NGAL, Laminin gamma1,5-Lipoxygenase, Laminin I, LXR alpha/NR1H3, Laminin S, LXR beta/NR1H2,Laminin-1, Livin, Laminin-5, LEX, LAMP, LMIR1/CD300A, Langerin,LMIR2/CD300c, LAR, LMIR3/CD300LF, Latexin, LMIR5/CD300LB, Layilin,LMIR6/CD300LE, LBP, LMO2, LDL R, LOX-1/SR-E1, LECT2, LRH-1/NR5A2, LEDGF,LRIG, Lefty, LRIG3, Lefty-1, LRP-I, Lefty-A, LRP-6, Legumain,LSECtin/CLEC4G, Leptin, Lumican, Leptin R, CXCL15/Lungkine, LeukotrieneB4, XCL1/Lymphotactin, Leukotriene B4 R1, Lymphotoxin, LEF, Lymphotoxinbeta/TNFSF3, LIF R alpha, Lymphotoxin beta R/TNFRSF3, LIGHT/TNFSF4, Lyn,Limitin, Lyp, LIMPII/SR-B2, Lysyl Oxidase Homolog 2, LIN-28, LYVE-I,LINGO-I, alpha 2-Macroglobulin, CXCL9/MIG, MAD2L1, Mimecan, MAdCAM-1,Mindin, MafB, Mineralocorticoid R/NR3C2, MafF, CCL3L1/MIP-1 alphaIsoform LD78 beta, MafG, CCL3/MIP-1 alpha, MafK, CCL4L1/LAG-1,MAG/Siglec-4-a, CCL4/MIP-1 beta, MANF, CCL5/MEP-1 delta, MAP2,CCL9/10/MIP-1 gamma, MAPK, MIP-2, Marapsin/Pancreasin, CCL19/MIP-3 beta,MARCKS, CCL20/MIP-3 alpha, MARCO, MIP-I, Mashl, MIP-II, Matrilin-2,MIP-III, Matrilin-3, MIS/AMH, Matrilin-4, MIS RII, Matriptase/ST14,MIXL1, MBL, MKK3/MKK6, MBL-2, MKK3, Melanocortin 3R/MC3R, MKK4,MCAM/CD146, MKK6, MCK-2, MKK7, McI-I, MKP-3, MCP-6, MLH-I, CCL2/MCP-1,MLK4 alpha, MCP-11, MMP, CCL8/MCP-2, MMP-1, CCL7/MCP-3/MARC, MMP-2,CCL13/MCP-4, MMP-3, CCL2/MCP-5, MMP-7, M-CSF, MMP-8, M-CSF R, MMP-9,MCV-type II, MMP-IO, MD-I, MMP-I 1, MD-2, MMP-12, CCL22/MDC, MMP-13,MDL-1/CLEC5A, MMP-14, MDM2, MMP-15, MEA-I, MMP-16/MT3-MMP, MEK1/MEK2,MMP-24/MT5-MMP, MEK1, MMP-25/MT6-MMP, MEK2, MMP-26, Melusin, MMR, MEPE,MOG, Meprin alpha, CCL23/MPIF-1, Meprin beta, M-Ras/R-Ras3, Mer, Mrel 1,Mesothelin, MRPI Meteorin, MSK1/MSK2, Methionine Aminopeptidase 1, MSK1,Methionine Aminopeptidase, MSK2, Methionine Aminopeptidase 2, MSP,MFG-E8, MSP R/Ron, MFRP, Mug, MgcRacGAP, MULT-I, MGL2, Musashi-1, MGMT,Musashi-2, MIA, MuSK, MICA, MutY DNA Glycosylase, MICB, MyD88,MICUICLEC12A, Myeloperoxidase, beta 2 Microglobulin, Myocardin, Midkine,Myocilin, MIF, Myoglobin, NAIP NGFI-B gamma/NR4A3, Nanog, NgR2/NgRH1,CXCL7/NAP-2, NgR3/NgRH2, Nbsl, Nidogen-1/Entactin, NCAM-1/CD56,Nidogen-2, NCAM-L1, Nitric Oxide, Nectin-1, Nitrotyrosine, Nectin-2/CD112, NKG2A, Nectin-3, NKG2C, Nectin-4, NKG2D, Neogenin, NKp30,Neprilysin/CDIO, NKp44, Neprilysin-2/MMEL1/MMEL2, NKp46/NCR, Nestin,NKp80/KLRF, NETO2, NKX2.5, Netrin-1, NMDA R, NR1 Subunit, Netrin-2, NMDAR, NR2A Subunit, Netrin-4, NMDA R, NR2B Subunit, Netrin-Gla, NMDA R,NR2C Subunit, Netrin-G2a, N-Me-6,7-diOH-TIQ, Neuregulin-1/NRG1, Nodal,Neuregulin-3/NRG3, Noggin, Neuritin, Nogo Receptor, NeuroDi, Nogo-A,Neurofascin, NOMO, Neurogenin-1, Nope, Neurogenin-2, Norrin,Neurogenin-3, eNOS, Neurolysin, iNOS, Neurophysin II, nNOS,Neuropilin-1, Notch-1, Neuropilin-2, Notch-2, Neuropoietin, Notch-3,Neurotrimin, Notch-4, Neurturin, NOV/CCN3, NFAMi, NRAGE, NF-H, NrCAM,NFkB1, NRL, NFkB2, NT-3, NF-L, NT-4, NF-M, NTB-A/SLAMF6, NG2/MCSP, NTH1,NGF RITNFRSF6, Nucleostemin, beta-NGF, Nurr-1/NR4A2, NGFI-B alpha/NR4A1,OAS2, Orexin B, OBCAM, OSCAR, OCAM, OSF-2/Periostin, OCIIJCLEC2d,Oncostatin M/OSM, OCILRP2/CLEC21, OSM R beta, Oct-3/4,Osteoactivin/GPNMB, OGG1, Osteoadherin, Olig 1, 2, 3, Osteocalcin,Olig1, Osteocrin, Olig2, Osteopontin, Olig3, OsteoprotegeriniTNFRSFi IB,Oligodendrocyte Marker 01, Otx2, Oligodendrocyte Marker 04, OV-6, OMgp,OX40ITNFRSF4, Opticin, OX40 LigandfTNFSF4, Orexin A, OAS2, Orexin B,OBCAM, OSCAR, OCAM, OSF-2/Periostin, OCIIJCLEC2d, Oncostatin M/OSM,OCILRP2/CLEC2i, OSM R beta, Oct-3/4, Osteoactivin/GPNMB, OGG1,Osteoadherin, Olig 1, 2, 3, Osteocalcin, Olig1, Osteocrin, Olig2,Osteopontin, Olig3, Osteoprotegerin/TNFRSF1 IB, Oligodendrocyte Marker01, Otx2, Oligodendrocyte Marker 04, OV-6, OMgp, OX40/TNFRSF4, Opticin,OX40 Ligand/TNFSF4, Orexin A, RACK1, Ret, Rad1, REV-ERB alpha/NR1D1,Rad17, REV-ERB beta/NR1D2, Rad51, Rex-1, Rae-1, RGM-A, Rae-1 alpha,RGM-B, Rae-1 beta, RGM-C, Rae-1 delta, Rheb, Rae-1 epsilon, RibosomalProtein S6, Rae-1 gamma, RIP1, Raf-1, ROBO1, RAGE, ROBO2, Ra1A/Ra1B,R0B03, RaIA, ROBO4, RaIB, R0R/NRIF1-3 (pan), RANK/TNFRSF1 1A, RORalpha/NR1F1, CCL5/RANTES, ROR gamma/NR1F3, Rap1A/B, RTK-like OrphanReceptor 1/ROR1, RAR alpha/NR1B1, RTK-like Orphan Receptor 2/ROR2, RARbeta/NR1B2, RP105, RAR gamma/NR1B3, RP A2, Ras, RSK (pan), RBP4,RSK1/RSK2, RECK, RSK1, Reg 2/PAP, RSK2, Reg I, RSK3, Reg II, RSK4, RegIII, R-Spondin 1, Reg I1ia, R-Spondin 2, Reg IV, R-Spondin 3, Relaxin-1,RUNX1/CBFA2, Relaxin-2, RUNX2/CBFA1, Relaxin-3, RUNX3/CBFA3, RELM alpha,RXR alpha/NR2B1, RELM beta, RXR beta/NR2B2, RELT/TNFRSF19L, RXRgamma/NR2B3, Resistin, S1OOAlO, SLITRK5, S100A8, SLPI, S100A9,SMAC/Diablo, S1OOB, Smad1, S1OOP, Smad2, SALL1, Smad3,delta-Sarcoglycan, Smad4, Sca-1/Ly6, Smad5, SCD-I, Smad7, SCF, Smad8,SCF R/c-kit, SMC1, SCGF, alpha-Smooth Muscle Actin, SCI/Tall, SMUG1,SCP3/SYCP3, Snail, CXCL12/SDF-1, Sodium Calcium Exchanger 1,SDNSF/MCFD2, Soggy-1, alpha-Secretase, Sonic Hedgehog, gamma-Secretase,S or CS1, beta-Secretase, S or CS3, E-Selectin, Sortilin, L-Selectin,SOST, P-Selectin, SOX1, Semaphorin 3A, SOX2, Semaphorin 3C, SOX3,Semaphorin 3E, SOX7, Semaphorin 3F, SOX9, Semaphorin 6A, SOX10,Semaphorin 6B, SOX 17, Semaphorin 6C, SOX21 Semaphorin 6D, SPARC,Semaphorin 7 A, SPARC-like 1, Separase, SP-D, SerineThreoninePhosphatase Substrate I, Spinesin, Serpin A1, F-Spondin, Serpin A3,SR-AI/MSR, Serpin A4/Kallistatin, Src, Serpin AS/Protein C Inhibitor,SREC-I/SR-F1, Serpin A8/Angiotensinogen, SREC-II, Serpin B5, SSEA-I,Serpin C1/Antithrombin-III, SSEA-3, Serpin D1/Heparin Cofactor II,SSEA-4, Serpin E1/PAI-1, ST7/LRP12, Serpin E2, Stabilin-1, Serpin F1,Stabilin-2, Serpin F2, Stanniocalcin 1, Serpin G1/C1 Inhibitor,Stanniocalcin 2, Serpin 12, STAT1, Serum Amyloid A1, STAT2, SF-1/NR5A1,STAT3, SGK, STAT4, SHBG, STAT5a/b, SHIP, STAT5a, SHP/NROB2, STAT5b,SHP-I, STATE, SHP-2, VE-Statin, SIGIRR, Stella/Dppa3, Siglec-2/CD22,STRO-I, Siglec-3/CD33, Substance P, Siglec-5, Sulfamidase/SGSH,Siglec-6, Sulfatase Modifying Factor 1/SUMF1, Siglec-7, SulfataseModifying Factor 2/SUMF2, Siglec-9, SUMO1, Siglec-10, SUMO2/3/4,Siglec-11, SUMO3, Siglec-F, Superoxide Dismutase, SIGNR1/CD209,Superoxide Dismutase-1/Cu[0099]—Zn SOD, SIGNR4, SuperoxideDismutase-2/Mn-SOD, SIRP beta 1, Superoxide Dismutase-3/EC-SOD, SK1,Survivin, SLAM/CD150, Synapsin I, Sleeping Beauty Transposase,Syndecan-I/CD 138, Slit3, Syndecan-2, SLITRK1, Syndecan-3, SLITRK2,Syndecan-4, SLITRK4, TACITNFRSF13B, TMEFF 1lTomoregulin-1, TAO2, TMEFF2,TAPPI, TNF-alpha/TNFSF IA, CCL17/TARC, TNF-betaITNFSF1B, Tau, TNFR1/TNFRSFIA, TC21/R-Ras2, TNF RII/TNFRSF1B, TCAM-I, TOR, TCCR/WSX-1,TP-I, TC-PTP, TP63/TP73L, TDG, TR, CCL25/TECK, TR alpha/NR1A1, TenascinC, TR beta 1/NR1A2, Tenascin R, TR2/NR2C1, TER-119, TR4/NR2C2, TERT,TRA-1-85, Testican 1/SPOCK1, TRADD, Testican 2/SPOCK2,TRAF-1, Testican3/SPOCK3, TRAF-2, TFPI, TRAF-3, TFPI-2, TRAF-4, TGF-alpha, TRAF-6,TGF-beta, TRAILITNFSF10, TGF-beta 1, TRAIL R1/TNFRSFIOA, LAP (TGF-beta1), TRAIL R2ITNFRSF10B, Latent TGF-beta 1, TRAIL R3/TNFRSF10C, TGF-beta1.2, TRAIL R4/TNFRSF10D, TGF-beta 2, TRANCEITNFSF1 1, TGF-beta 3, TfR(Transferrin R), TGF-beta 5, Apo-Transferrin, Latent TGF-beta by 1,Holo-Transferrin, Latent TGF-beta bp2, Trappin-2/Elafin, Latent TGF-betabp4, TREM-1, TGF-beta R1/ALK-5, TREM-2, TGF-beta R11, TREM-3, TGF-betaRIIb, TREML1/TLT-1, TGF-beta RIII, TRF-I, Thermolysin, TRF-2,Thioredoxin-1, TRH-degrading Ectoenzyme/TRHDE, Thioredoxin-2, TRIMS,Thioredoxin-80, Tripeptidyl-Peptidase I, Thioredoxin-like 5/TRP14, TrkA,THOP1, TrkB, Thrombomodulin/CD141, TrkC, Thrombopoietin, TROP-2,Thrombopoietin R, Troponin I Peptide 3, Thrombospondin-1, Troponin T,Thrombospondin-2, TROY/TNFRSF 19, Thrombospondin-4, Trypsin 1,Thymopoietin, Trypsin 2/PRSS2, Thymus Chemokine-1, Trypsin 3/PRSS3,Tie-1, Tryptase-5/Prss32, Tie-2, Tryptase alpha/TPS1, TIM-I/KIM-I/HAVCR,Tryptase beta-1/MCPT-7, TIM-2, Tryptase beta-2/TPSB2, TIM-3, Tryptaseepsilon/BSSP-4, TIM-4, Tryptase gamma-1/TPSG1, TIM-5, TryptophanHydroxylase, TIM-6, TSC22, TIMP-I, TSG, TIMP-2, TSG-6, TIMP-3, TSK,TIMP-4, TSLP, TL1A/TNFSF15, TSLP R, TLR1, TSP50, TLR2, beta-III Tubulin,TLR3, TWEAK/TNFSF12, TLR4, TWEAK R/TNFRSF 12, TLR5, Tyk2, TLR6,Phospho-Tyrosine, TLR9, Tyrosine Hydroxylase, TLX/NR2E1, TyrosinePhosphatase Substrate I, Ubiquitin, UNC5H3, Ugi, UNC5H4, UGRP1, UNG,ULBP-I, uPA, ULBP-2, uPAR, ULBP-3, URB, UNC5H1, UVDE, UNC5H2, VanilloidR1, VEGF R, VASA, VEGF R1/Flt-1, Vasohibin, VEGF R2/KDR/Flk-1, Vasorin,VEGF R3/FU-4, Vasostatin, Versican, Vav-1, VG5Q, VCAM-1, VHR, VDR/NR1I1,Vimentin, VEGF, Vitronectin, VEGF-B, VLDLR, VEGF-C, vWF-A2, VEGF-D,Synuclein-alpha, Ku70, WASP, Wnt-7b, WIF-I, Wnt-8a WISP-1/CCN4, Wnt-8b,WNK1, Wnt-9a, Wnt-1, Wnt-9b, Wnt-3a, Wnt-10a, Wnt-4, Wnt-10b, Wnt-5a,Wnt-1, Wnt-5b, wnvNS3, Wnt7a, XCR1, XPE/DDB1, XEDAR, XPE/DDB2, Xg, XPF,XIAP, XPG, XPA, XPV, XPD, XRCC1, Yes, YY1, EphA4.

Numerous human ion channels are targets of particular interest.Non-limiting examples include 5-hydroxytryptamine 3 receptor B subunit,5-hydroxytryptamine 3 receptor precursor, 5-hydroxytryptamine receptor 3subunit C, AAD 14 protein, Acetylcholine receptor protein, alpha subunitprecursor, Acetylcholine receptor protein, beta subunit precursor,Acetylcholine receptor protein, delta subunit precursor, Acetylcholinereceptor protein, epsilon subunit precursor, Acetylcholine receptorprotein, gamma subunit precursor, Acid sensing ion channel 3 splicevariant b, Acid sensing ion channel 3 splice variant c, Acid sensing ionchannel 4, ADP-ribose pyrophosphatase, mitochondrial precursor, Alpha1A-voltage-dependent calcium channel, Amiloride-sensitive cation channel1, neuronal, Amiloride-sensitive cation channel 2, neuronalAmiloride-sensitive cation channel 4, isoform 2, Amiloride-sensitivesodium channel, Amiloride-sensitive sodium channel alpha-subunit,Amiloride-sensitive sodium channel beta-subunit, Amiloride-sensitivesodium channel delta-subunit, Amiloride-sensitive sodium channelgamma-subunit, Annexin A7, Apical-like protein, ATP-sensitive inwardrectifier potassium channel 1, ATP-sensitive inward rectifier potassiumchannel 10, ATP-sensitive inward rectifier potassium channel 11,ATP-sensitive inward rectifier potassium channel 14, ATP-sensitiveinward rectifier potassium channel 15, ATP-sensitive inward rectifierpotassium channel 8, Calcium channel alpha12.2 subunit, Calcium channelalpha12.2 subunit, Calcium channel alpha1E subunit, delta19 delta40delta46 splice variant, Calcium-activated potassium channel alphasubunit 1, Calcium-activated potassium channel beta subunit 1,Calcium-activated potassium channel beta subunit 2, Calcium-activatedpotassium channel beta subunit 3, Calcium-dependent chloride channel-1,Cation channel TRμM4B, CDNA FLJ90453 fis, clone NT2RP3001542, highlysimilar to Potassium channel tetramerisation domain containing 6, CDNAFL190663 fis, clone PLACE 1005031, highly similar to Chlorideintracellular channel protein 5, CGMP-gated cation channel beta subunit,Chloride channel protein, Chloride channel protein 2, Chloride channelprotein 3, Chloride channel protein 4, Chloride channel protein 5,Chloride channel protein 6, Chloride channel protein C1C-Ka, Chloridechannel protein C C-Kb, Chloride channel protein, skeletal muscle,Chloride intracellular channel 6, Chloride intracellular channel protein3, Chloride intracellular channel protein 4, Chloride intracellularchannel protein 5, CHRNA3 protein, Clcn3e protein, CLCNKB protein, CNGA4protein, Cullin-5, Cyclic GMP gated potassium channel,Cyclic-nucleotide-gated cation channel 4, Cyclic-nucleotide-gated cationchannel alpha 3, Cyclic-nucleotide-gated cation channel beta 3,Cyclic-nucleotide-gated olfactory channel, Cystic fibrosis transmembraneconductance regulator, Cytochrome B-245 heavy chain,Dihydropyridine-sensitive L-type, calcium channel alpha-2/delta subunitsprecursor, FXYD domain-containing ion transport regulator 3 precursor,FXYD domain-containing ion transport regulator 5 precursor, FXYDdomain-containing ion transport regulator 6 precursor, FXYDdomain-containing ion transport regulator 7, FXYD domain-containing iontransport regulator 8 precursor, G protein-activated inward rectifierpotassium channel 1, G protein-activated inward rectifier potassiumchannel 2, G protein-activated inward rectifier potassium channel 3, Gprotein-activated inward rectifier potassium channel 4,Gamma-aminobutyric-acid receptor alpha-1 subunit precursor,Gamma-aminobutyric-acid receptor alpha-2 subunit precursor,Gamma-aminobutyric-acid receptor alpha-3 subunit precursor,Gamma-aminobutyric-acid receptor alpha-4 subunit precursor,Gamma-aminobutyric-acid receptor alpha-5 subunit precursor,Gamma-aminobutyric-acid receptor alpha-6 subunit precursor,Gamma-aminobutyric-acid receptor beta-1 subunit precursor,Gamma-aminobutyric-acid receptor beta-2 subunit precursor,Gamma-aminobutyric-acid receptor beta-3 subunit precursor,Gamma-aminobutyric-acid receptor delta subunit precursor,Gamma-aminobutyric-acid receptor epsilon subunit precursor,Gamma-aminobutyric-acid receptor gamma-1 subunit precursor,Gamma-aminobutyric-acid receptor gamma-3 subunit precursor,Gamma-aminobutyric-acid receptor pi subunit precursor,Gamma-aminobutyric-acid receptor rho-1 subunit precursor,Gamma-aminobutyric-acid receptor rho-2 subunit precursor,Gamma-aminobutyric-acid receptor theta subunit precursor, GluR6 kainatereceptor, Glutamate receptor 1 precursor, Glutamate receptor 2precursor, Glutamate receptor 3 precursor, Glutamate receptor 4precursor, Glutamate receptor 7, Glutamate receptor B, Glutamatereceptor delta-1 subunit precursor, Glutamate receptor, ionotropickainate 1 precursor, Glutamate receptor, ionotropic kainate 2 precursor,Glutamate receptor, ionotropic kainate 3 precursor, Glutamate receptor,ionotropic kainate 4 precursor, Glutamate receptor, ionotropic kainate 5precursor, Glutamate [NMDA] receptor subunit 3A precursor, Glutamate[NMDA] receptor subunit 3B precursor, Glutamate [NMDA] receptor subunitepsilon 1 precursor, Glutamate [NMDA] receptor subunit epsilon 2precursor, Glutamate [NMDA] receptor subunit epsilon 4 precursor,Glutamate [NMDA] receptor subunit zeta 1 precursor, Glycine receptoralpha-1 chain precursor, Glycine receptor alpha-2 chain precursor,Glycine receptor alpha-3 chain precursor, Glycine receptor beta chainprecursor, H/ACA ribonucleoprotein complex subunit 1, High affinityimmunoglobulin epsilon receptor beta-subunit, Hypothetical proteinDKFZp31310334, Hypothetical protein DKFZp761M1724, Hypothetical proteinFLJ12242, Hypothetical protein FLJ14389, Hypothetical protein FLJ14798,Hypothetical protein FLJ14995, Hypothetical protein FLJ16180,Hypothetical protein FLJ16802, Hypothetical protein FLJ32069,Hypothetical protein FLJ37401, Hypothetical protein FLJ38750,Hypothetical protein FLJ40162, Hypothetical protein FLJ41415,Hypothetical protein FLJ90576, Hypothetical protein FLJ90590,Hypothetical protein FLJ90622, Hypothetical protein KCTD15, Hypotheticalprotein MGC15619, Inositol 1,4,5-trisphosphate receptor type 1, Inositol1,4,5-trisphosphate receptor type 2, Inositol 1,4,5-trisphosphatereceptor type 3, Intermediate conductance calcium-activated potassiumchannel protein 4, Inward rectifier potassium channel 13, Inwardrectifier potassium channel 16, Inward rectifier potassium channel 4,Inward rectifying K(+) channel negative regulator Kir2.2v, Kainatereceptor subunit KA2a, KCNH5 protein, KCTD 17 protein, KCTD2 protein,Keratinocytes associated transmembrane protein 1, Kv channel-interactingprotein 4, Melastatin 1, Membrane protein MLC1, MGC 15619 protein,Mucolipin-1, Mucolipin-2, Mucolipin-3, Multidrug resistance-associatedprotein 4, N-methyl-D-aspartate receptor 2C subunit precursor, NADPHoxidase homolog 1, Nay 1.5, Neuronal acetylcholine receptor protein,alpha-10 subunit precursor, Neuronal acetylcholine receptor protein,alpha-2 subunit precursor, Neuronal acetylcholine receptor protein,alpha-3 subunit precursor, Neuronal acetylcholine receptor protein,alpha-4 subunit precursor, Neuronal acetylcholine receptor protein,alpha-5 subunit precursor, Neuronal acetylcholine receptor protein,alpha-6 subunit precursor, Neuronal acetylcholine receptor protein,alpha-7 subunit precursor, Neuronal acetylcholine receptor protein,alpha-9 subunit precursor, Neuronal acetylcholine receptor protein,beta-2 subunit precursor, Neuronal acetylcholine receptor protein,beta-3 subunit precursor, Neuronal acetylcholine receptor protein,beta-4 subunit precursor, Neuronal voltage-dependent calcium channelalpha 2D subunit, P2X purinoceptor 1, P2X purinoceptor 2, P2Xpurinoceptor 3, P2X purinoceptor 4, P2X purinoceptor 5, P2X purinoceptor6, P2X purinoceptor 7, Pancreatic potassium channel TALK-Ib, Pancreaticpotassium channel TALK-Ic, Pancreatic potassium channel TALK-Id,Phospholemman precursor, Plasmolipin, Polycystic kidney disease 2related protein, Polycystic kidney disease 2-like 1 protein, Polycystickidney disease 2-like 2 protein, Polycystic kidney disease and receptorfor egg jelly related protein precursor, Polycystin-2, Potassium channelregulator, Potassium channel subfamily K member 1, Potassium channelsubfamily K member 10, Potassium channel subfamily K member 12,Potassium channel subfamily K member 13, Potassium channel subfamily Kmember 15, Potassium channel subfamily K member 16, Potassium channelsubfamily K member 17, Potassium channel subfamily K member 2, Potassiumchannel subfamily K member 3, Potassium channel subfamily K member 4,Potassium channel subfamily K member 5, Potassium channel subfamily Kmember 6, Potassium channel subfamily K member 7, Potassium channelsubfamily K member 9, Potassium channel tetramerisation domaincontaining 3, Potassium channel tetramerisation domain containingprotein 12, Potassium channel tetramerisation domain containing protein14, Potassium channel tetramerisation domain containing protein 2,Potassium channel tetramerisation domain containing protein 4, Potassiumchannel tetramerisation domain containing protein 5, Potassium channeltetramerization domain containing 10, Potassium channel tetramerizationdomain containing protein 13, Potassium channel tetramerizationdomain-containing 1, Potassium voltage-gated channel subfamily A member1, Potassium voltage-gated channel subfamily A member 2, Potassiumvoltage-gated channel subfamily A member 4, Potassium voltage-gatedchannel subfamily A member 5, Potassium voltage-gated channel subfamilyA member 6, Potassium voltage-gated channel subfamily B member 1,Potassium voltage-gated channel subfamily B member 2, Potassiumvoltage-gated channel subfamily C member 1, Potassium voltage-gatedchannel subfamily C member 3, Potassium voltage-gated channel subfamilyC member 4, Potassium voltage-gated channel subfamily D member 1,Potassium voltage-gated channel subfamily D member 2, Potassiumvoltage-gated channel subfamily D member 3, Potassium voltage-gatedchannel subfamily E member 1, Potassium voltage-gated channel subfamilyE member 2, Potassium voltage-gated channel subfamily E member 3,Potassium voltage-gated channel subfamily E member 4, Potassiumvoltage-gated channel subfamily F member 1, Potassium voltage-gatedchannel subfamily G member 1, Potassium voltage-gated channel subfamilyG member 2, Potassium voltage-gated channel subfamily G member 3,Potassium voltage-gated channel subfamily G member 4, Potassiumvoltage-gated channel subfamily H member 1, Potassium voltage-gatedchannel subfamily H member 2, Potassium voltage-gated channel subfamilyH member 3, Potassium voltage-gated channel subfamily H member 4,Potassium voltage-gated channel subfamily H member 5, Potassiumvoltage-gated channel subfamily H member 6, Potassium voltage-gatedchannel subfamily H member 7, Potassium voltage-gated channel subfamilyH member 8, Potassium voltage-gated channel subfamily KQT member 1,Potassium voltage-gated channel subfamily KQT member 2, Potassiumvoltage-gated channel subfamily KQT member 3, Potassium voltage-gatedchannel subfamily KQT member 4, Potassium voltage-gated channelsubfamily KQT member 5, Potassium voltage-gated channel subfamily Smember 1, Potassium voltage-gated channel subfamily S member 2,Potassium voltage-gated channel subfamily S member 3, Potassiumvoltage-gated channel subfamily V member 2, Potassium voltage-gatedchannel, subfamily H, member 7, isoform 2, Potassium/sodiumhyperpolarization-activated cyclic nucleotide-gated channel 1,Potassium/sodium hyperpolarization-activated cyclic nucleotide-gatedchannel 2, Potassium/sodium hyperpolarization-activated cyclicnucleotide-gated channel 3, Potassium/sodium hyperpolarization-activatedcyclic nucleotide-gated channel 4, Probable mitochondrial importreceptor subunit TOM40 homolog, Purinergic receptor P2X5, isoform A,Putative 4 repeat voltage-gated ion channel, Putative chloride channelprotein 7, Putative GluR6 kainate receptor, Putative ion channel proteinCATSPER2 variant 1, Putative ion channel protein CATSPER2 variant 2,Putative ion channel protein CATSPER2 variant 3, Putative regulator ofpotassium channels protein variant 1, Putative tyrosine-proteinphosphatase TPTE, Ryanodine receptor 1, Ryanodine receptor 2, Ryanodinereceptor 3, SH3 KBP1 binding protein 1, Short transient receptorpotential channel 1, Short transient receptor potential channel 4, Shorttransient receptor potential channel 5, Short transient receptorpotential channel 6, Short transient receptor potential channel 7, Smallconductance calcium-activated potassium channel protein 1, Smallconductance calcium-activated potassium channel protein 2, isoform b,Small conductance calcium-activated potassium channel protein 3, isoformb, Small-conductance calcium-activated potassium channel SK2,Small-conductance calcium-activated potassium channel SK3, Sodiumchannel, Sodium channel beta-1 subunit precursor, Sodium channel proteintype II alpha subunit, Sodium channel protein type III alpha subunit,Sodium channel protein type IV alpha subunit, Sodium channel proteintype IX alpha subunit, Sodium channel protein type V alpha subunit,Sodium channel protein type VII alpha subunit, Sodium channel proteintype VIII alpha subunit, Sodium channel protein type X alpha subunit,Sodium channel protein type XI alpha subunit, Sodium- andchloride-activated ATP-sensitive potassium channel,Sodium/potassium-transporting ATPase gamma chain, Sperm-associatedcation channel 1, Sperm-associated cation channel 2, isoform 4,Syntaxin-1B1, Transient receptor potential cation channel subfamily Amember 1, Transient receptor potential cation channel subfamily M member2, Transient receptor potential cation channel subfamily M member 3,Transient receptor potential cation channel subfamily M member 6,Transient receptor potential cation channel subfamily M member 7,Transient receptor potential cation channel subfamily V member 1,Transient receptor potential cation channel subfamily V member 2,Transient receptor potential cation channel subfamily V member 3,Transient receptor potential cation channel subfamily V member 4,Transient receptor potential cation channel subfamily V member 5,Transient receptor potential cation channel subfamily V member 6,Transient receptor potential channel 4 epsilon splice variant, Transientreceptor potential channel 4 zeta splice variant, Transient receptorpotential channel 7 gamma splice variant, Tumor necrosis factor,alpha-induced protein 1, endothelial, Two-pore calcium channel protein2, VDAC4 protein, Voltage gated potassium channel Kv3.2b, Voltage gatedsodium channel beta1B subunit, Voltage-dependent anion channel,Voltage-dependent anion channel 2, Voltage-dependent anion-selectivechannel protein 1, Voltage-dependent anion-selective channel protein 2,Voltage-dependent anion-selective channel protein 3, Voltage-dependentcalcium channel gamma-1 subunit, Voltage-dependent calcium channelgamma-2 subunit, Voltage-dependent calcium channel gamma-3 subunit,Voltage-dependent calcium channel gamma-4 subunit, Voltage-dependentcalcium channel gamma-5 subunit, Voltage-dependent calcium channelgamma-6 subunit, Voltage-dependent calcium channel gamma-7 subunit,Voltage-dependent calcium channel gamma-8 subunit, Voltage-dependentL-type calcium channel alpha-1C subunit, Voltage-dependent L-typecalcium channel alpha-1D subunit, Voltage-dependent L-type calciumchannel alpha-IS subunit, Voltage-dependent L-type calcium channelbeta-1 subunit, Voltage-dependent L-type calcium channel beta-2 subunit,Voltage-dependent L-type calcium channel beta-3 subunit,Voltage-dependent L-type calcium channel beta-4 subunit,Voltage-dependent N-type calcium channel alpha-1B subunit,Voltage-dependent P/Q-type calcium channel alpha-1A subunit,Voltage-dependent R-type calcium channel alpha-1E subunit,Voltage-dependent T-type calcium channel alpha-1G subunit,Voltage-dependent T-type calcium channel alpha-1H subunit,Voltage-dependent T-type calcium channel alpha-1I subunit, Voltage-gatedL-type calcium channel alpha-1 subunit, Voltage-gated potassium channelbeta-1 subunit, Voltage-gated potassium channel beta-2 subunit,Voltage-gated potassium channel beta-3 subunit, Voltage-gated potassiumchannel KCNA7. The Nav1.x family of human voltage-gated sodium channelsis also a particularly promising target. This family includes, forexample, channels Nav1.6 and Nav1.8.

In certain embodiments, the therapeutic protein may be a G-ProteinCoupled Receptor (GPCR). Exemplary GPCRs include, but are not limitedto, Class A Rhodopsin like receptors such as Muscatinic (Muse.)acetylcholine Vertebrate type 1, Muse, acetylcholine Vertebrate type 2,Muse, acetylcholine Vertebrate type 3, Muse, acetylcholine Vertebratetype 4; Adrenoceptors (Alpha Adrenoceptors type 1, Alpha Adrenoceptorstype 2, Beta Adrenoceptors type 1, Beta Adrenoceptors type 2, BetaAdrenoceptors type 3, Dopamine Vertebrate type 1, Dopamine Vertebratetype 2, Dopamine Vertebrate type 3, Dopamine Vertebrate type 4,Histamine type 1, Histamine type 2, Histamine type 3, Histamine type 4,Serotonin type 1, Serotonin type 2, Serotonin type 3, Serotonin type 4,Serotonin type 5, Serotonin type 6, Serotonin type 7, Serotonin type 8,other Serotonin types, Trace amine, Angiotensin type 1, Angiotensin type2, Bombesin, Bradykinin, C5a anaphylatoxin, Fmet-leu-phe, APJ like,Interleukin-8 type A, Interleukin-8 type B, Interleukin-8 type others,C-C Chemokine type 1 through type 11 and other types, C-X-C Chemokine(types 2 through 6 and others), C-X3-C Chemokine, Cholecystokinin CCK,CCK type A, CCK type B, CCK others, Endothelin, Melanocortin (Melanocytestimulating hormone, Adrenocorticotropic hormone, Melanocortin hormone),Duffy antigen, Prolactin-releasing peptide (GPRIO), Neuropeptide Y (type1 through 7), Neuropeptide Y, Neuropeptide Y other, Neurotensin, Opioid(type D, K, M, X), Somatostatin (type 1 through 5), Tachykinin(Substance P (NK1), Substance K (NK2), Neuromedin K (NK3), Tachykininlike 1, Tachykinin like 2, Vasopressin/vasotocin (type 1 through 2),Vasotocin, Oxytocin/mesotocin, Conopressin, Galanin like,Proteinase-activated like, Orexin & neuropeptides FF.QRFP, Chemokinereceptor-like, Neuromedin U like (Neuromedin U, PRXamide), hormoneprotein (Follicle stimulating hormone, Lutropin-choriogonadotropichormone, Thyrotropin, Gonadotropin type I, Gonadotropin type II),(Rhod)opsin, Rhodopsin Vertebrate (types 1-5), Rhodopsin Vertebrate type5, Rhodopsin Arthropod, Rhodopsin Arthropod type 1, Rhodopsin Arthropodtype 2, Rhodopsin Arthropod type 3, Rhodopsin Mollusc, Rhodopsin,Olfactory (Olfactory II fam 1 through 13), Prostaglandin (prostaglandinE2 subtype EP1, Prostaglandin E2/D2 subtype EP2, prostaglandin E2subtype EP3, Prostaglandin E2 subtype EP4, Prostaglandin F2-alpha,Prostacyclin, Thromboxane, Adenosine type 1 through 3, Purinoceptors,Purinoceptor P2RY1-4,6,1 1 GPR91, Purinoceptor P2RY5,8,9,10GPR35,92,174, Purinoceptor P2RY12-14 GPR87 (UDP-Glucose), Cannabinoid,Platelet activating factor, Gonadotropin-releasing hormone,Gonadotropin-releasing hormone type I, Gonadotropin-releasing hormonetype II, Adipokinetic hormone like, Corazonin, Thyrotropin-releasinghormone & Secretagogue, Thyrotropin-releasing hormone, Growth hormonesecretagogue, Growth hormone secretagogue like, Ecdysis-triggeringhormone (ETHR), Melatonin, Lysosphingolipid & LPA (EDG), Sphingosine1-phosphate Edg-1, Lysophosphatidic acid Edg-2, Sphingosine 1-phosphateEdg-3, Lysophosphatidic acid Edg-4, Sphingosine 1-phosphate Edg-5,Sphingosine 1-phosphate Edg-6, Lysophosphatidic acid Edg-7, Sphingosine1-phosphate Edg-8, Edg Other Leukotriene B4 receptor, Leukotriene B4receptor BLT1, Leukotriene B4 receptor BLT2, Class A Orphan/other,Putative neurotransmitters, SREB, Mas proto-oncogene & Mas-related(MRGs), GPR45 like, Cysteinyl leukotriene, G-protein coupled bile acidreceptor, Free fatty acid receptor (GP40, GP41, GP43), Class B Secretinlike, Calcitonin, Corticotropin releasing factor, Gastric inhibitorypeptide, Glucagon, Growth hormone-releasing hormone, Parathyroidhormone, PACAP, Secretin, Vasoactive intestinal polypeptide,Latrophilin, Latrophilin type 1, Latrophilin type 2, Latrophilin type 3,ETL receptors, Brain-specific angiogenesis inhibitor (BAI),Methuselah-like proteins (MTH), Cadherin EGF LAG (CELSR), Very largeG-protein coupled receptor, Class C Metabotropic glutamate/pheromone,Metabotropic glutamate group I through III, Calcium-sensing like,Extracellular calcium-sensing, Pheromone, calcium-sensing like other,Putative pheromone receptors, GABA-B, GABA-B subtype 1, GABA-B subtype2, GABA-B like, Orphan GPRC5, Orphan GPCR6, Bride of sevenless proteins(BOSS), Taste receptors (T1R), Class D Fungal pheromone, Fungalpheromone A-Factor like (STE2.STE3), Fungal pheromone B like(BAR,BBR,RCB,PRA), Class E cAMP receptors, Ocular albinism proteins,Frizzled/Smoothened family, frizzled Group A (Fz 1&2&4&5&7-9), frizzledGroup B (Fz 3 & 6), frizzled Group C (other), Vomeronasal receptors,Nematode chemoreceptors, Insect odorant receptors, and Class ZArchaeal/bacterial/fiingal opsins.

In certain embodiments, the serum albumin binding Fn3 fusions describedherein may comprise any of the following active polypeptides: BOTOX,Myobloc, Neurobloc, Dysport (or other serotypes of botulinumneurotoxins), alglucosidase alfa, daptomycin, YH-16, choriogonadotropinalfa, filgrastim, cetrorelix, interleukin-2, aldesleukin, teceleukin,denileukin diftitox, interferon alfa-n3 (injection), interferon alfa-nl,DL-8234, interferon, Suntory (gamma-Ia), interferon gamma, thymosinalpha 1, tasonermin, DigiFab, ViperaTAb, EchiTAb, CroFab, nesiritide,abatacept, alefacept, Rebif, eptoterminalfa, teriparatide(osteoporosis), calcitonin injectable (bone disease), calcitonin (nasal,osteoporosis), etanercept, hemoglobin glutamer 250 (bovine), drotrecoginalfa, collagenase, carperitide, recombinant human epidermal growthfactor (topical gel, wound healing), DWP-401, darbepoetin alfa, epoetinomega, epoetin beta, epoetin alfa, desirudin, lepirudin, bivalirudin,nonacog alpha, Mononine, eptacog alfa (activated), recombinant FactorVIII+VWF, Recombinate, recombinant Factor VIII, Factor VIII(recombinant), Alphanate, octocog alfa, Factor VIII, palifermin,Indikinase, tenecteplase, alteplase, pamiteplase, reteplase,nateplase.monteplase, follitropin alfa, rFSH, hpFSH, micafungin,pegfilgrastim, lenograstim, nartograstim, sermorelin, glucagon,exenatide, pramlintide, imiglucerase, galsulfase, Leucotropin,molgramostim, triptorelin acetate, histrelin (subcutaneous implant,Hydron), deslorelin, histrelin, nafarelin, leuprolide sustained releasedepot (ATRIGEL), leuprolide implant (DUROS), goserelin, somatropin,Eutropin, KP-102 program, somatropin, somatropin, mecasermin (growthfailure), enfuvirtide, Org-33408, insulin glargine, insulin glulisine,insulin (inhaled), insulin lispro, insulin detemir, insulin (buccal,RapidMist), mecasermin rinfabate, anakinra, celmoleukin, 99 mTc-apcitideinjection, myelopid, Betaseron, glatiramer acetate, Gepon, sargramostim,oprelvekin, human leukocyte-derived alpha interferons, Bilive, insulin(recombinant), recombinant human insulin, insulin aspart, mecasermin,Roferon-A, interferon-alpha 2, Alfaferone, interferon alfacon-1,interferon alpha, Avonex recombinant human luteinizing hormone, dornasealfa, trafermin, ziconotide, taltirelin, diboterminalfa, atosiban,becaplermin, eptifibatide, Zemaira, CTC-lll, Shanvac-B, HPV vaccine(quadrivalent), NOV-002, octreotide, lanreotide, ancestim, agalsidasebeta, agalsidase alfa, laronidase, prezatide copper acetate (topicalgel), rasburicase, ranibizumab, Actimmune, PEG-Intron, Tricomin,recombinant house dust mite allergy desensitization injection,recombinant human parathyroid hormone (PTH) 1-84 (sc, osteoporosis),epoetin delta, transgenic antithrombin III, Granditropin, Vitrase,recombinant insulin, interferon-alpha (oral lozenge), GEM-2 IS,vapreotide, idursulfase, omapatrilat, recombinant serum albumin,certolizumab pegol, glucarpidase, human recombinant C1 esteraseinhibitor (angioedema), lanoteplase, recombinant human growth hormone,enfuvirtide (needle-free injection, Biojector 2000), VGV-I, interferon(alpha), lucinactant, aviptadil (inhaled, pulmonary disease), icatibant,ecallantide, omiganan, Aurograb, pexiganan acetate, ADI-PEG-20, LDI-200,degarelix, cintredekin besudotox, FavId, MDX-1379, ISAtx-247,liraglutide, teriparatide (osteoporosis), tifacogin, AA-4500, T4N5liposome lotion, catumaxomab, DWP-413, ART-123, Chrysalin, desmoteplase,amediplase, corifollitropin alpha, TH-9507, teduglutide, Diamyd,DWP-412, growth hormone (sustained release injection), recombinantG-CSF, insulin (inhaled, AIR), insulin (inhaled, Technosphere), insulin(inhaled, AERx), RGN-303, DiaPep277, interferon beta (hepatitis C viralinfection (HCV)), interferon alfa-n3 (oral), belatacept, transdermalinsulin patches, AMG-531, MBP-8298, Xerecept, opebacan, AIDSVAX,GV-1001, LymphoScan, ranpirnase, Lipoxysan, lusupultide, MP52(beta-tricalciumphosphate carrier, bone regeneration), melanoma vaccine,sipuleucel-T, CTP-37, Insegia, vitespen, human thrombin (frozen,surgical bleeding), thrombin, TransMID, alfimeprase, Puricase,terlipressin (intravenous, hepatorenal syndrome), EUR-1008M, recombinantFGF-I (injectable, vascular disease), BDM-E, rotigaptide, ETC-216,P-113, MBI-594AN, duramycin (inhaled, cystic fibrosis), SCV-07, OPI-45,Endostatin, Angiostatin, ABT-5 10, Bowman Birk Inhibitor Concentrate,XMP-629, 99 mTc-Hynic-Annexin V, kahalalide F, CTCE-9908, teverelix(extended release), ozarelix, romidepsin, BAY-50-4798, interleukin-4,PRX-321, Pepscan, iboctadekin, rh lactoferrin, TRU-015, IL-21, ATN-161,cilengitide, Albuferon, Biphasix, IRX-2, omega interferon, PCK-3145,CAP-232, pasireotide, huN901-DM1, ovarian cancer immunotherapeuticvaccine, SB-249553, Oncovax-CL, OncoVax-P, BLP-25, CerVax-16,multi-epitope peptide melanoma vaccine (MART-I, gp 100, tyrosinase),nemifitide, rAAT (inhaled), rAAT (dermatological), CGRP (inhaled,asthma), pegsunercept, thymosin beta-4, plitidepsin, GTP-200,ramoplanin, GRASPA, OBI-I, AC-100, salmon calcitonin (oral, eligen),calcitonin (oral, osteoporosis), examorelin, capromorelin, Cardeva,velafermin, 131I-TM-601, KK-220, TP-10, ularitide, depelestat, hematide,Chrysalin (topical), rNAPc2, recombinant Factor VIII (PEGylatedliposomal), bFGF, PEGylated recombinant staphylokinase variant, V-10153,SonoLysis Prolyse, NeuroVax, CZEN-002, islet cell neogenesis therapy,rGLP-1, BIM-51077, LY-548806, exenatide (controlled release, Medisorb),AVE-0010, GA-GCB, avorelin, AOD-9604, linaclotide acetate, CETi-I,Hemospan, VAL (injectable), fast-acting insulin (injectable, Viadel),intranasal insulin, insulin (inhaled), insulin (oral, eligen),recombinant methionyl human leptin, pitrakinra subcutaneous injection,eczema), pitrakinra (inhaled dry powder, asthma), Multikine, RG-1068,MM-093, NBI-6024, AT-001, PI-0824, Org-39141, Cpn 10 (autoimmunediseases/inflammation), talactoferrin (topical), rEV-131 (ophthalmic),rEV-131 (respiratory disease), oral recombinant human insulin(diabetes), RPI-78M, oprelvekin (oral), CYT-99007 CTLA4-Ig, DTY-001,valategrast, interferon alfa-n3 (topical), IRX-3, RDP-58, Tauferon, bilesalt stimulated lipase, Merispase, alkaline phosphatase, EP-2104R,Melanotan-II, bremelanotide, ATL-104, recombinant human microplasmin,AX-200, SEMAX, ACV-I, Xen-2174, CJC-1008, dynorphin A, SI-6603, LABGHRH, AER-002, BGC-728, malaria vaccine (virosomes, PeviPRO), ALTU-135,parvovirus B 19 vaccine, influenza vaccine (recombinant neuraminidase),malaria/HBV vaccine, anthrax vaccine, Vacc-5q, Vacc-4x, HIV vaccine(oral), HPV vaccine, Tat Toxoid, YSPSL, CHS-13340, PTH(1-34) liposomalcream (Novasome), Ostabolin-C, PTH analog (topical, psoriasis),MBRI-93.02, MTB72F vaccine (tuberculosis), MVA-Ag85 A vaccine(tuberculosis), FAR-404, BA-210, recombinant plague F1V vaccine, AG-702,OxSODro1, rBetV1, Der-p1/Der-p2/Der-p7 allergen-targeting vaccine (dustmite allergy), PR1 peptide antigen (leukemia), mutant ras vaccine,HPV-16 E7 lipopeptide vaccine, labyrinthin vaccine (adenocarcinoma), CMLvaccine, WT1-peptide vaccine (cancer), IDD-5, CDX-110, Pentrys, Norelin,CytoFab, P-9808, VT-111, icrocaptide, telbermin (dermatological,diabetic foot ulcer), rupintrivir, reticulose, rGRF, P1A,alpha-galactosidase A, ACE-011, ALTU-140, CGX-1160, angiotensintherapeutic vaccine, D-4F, ETC-642, APP-018, rhMBL, SCV-07 (oral,tuberculosis), DRF-7295, ABT-828, ErbB2-specific immunotoxin(anticancer), DT388IL-3, TST-10088, PRO-1762, Combotox,cholecystokinin-B/gastrin-receptor binding peptides, 1 1 1ln-hEGF,AE-37, trastuzumab-DM1, Antagonist G, IL-12 (recombinant), μM-02734,IMP-321, rhIGF-BP3, BLX-883, CUV-1647 (topical), L-19 basedradioimmunotherapeutics (cancer), Re-188-P-2045, AMG-386, DC/I540/KLHvaccine (cancer), VX-001, AVE-9633, AC-9301, NY-ESO-I vaccine(peptides), NA17.A2 peptides, melanoma vaccine (pulsed antigentherapeutic), prostate cancer vaccine, CBP-501, recombinant humanlactoferrin (dry eye), FX-06, AP-214, WAP-8294A2 (injectable), ACP-HIP,SUN-11031, peptide YY [3-36] (obesity, intranasal), FGLL, atacicept,BR3-Fc, BN-003, BA-058, human parathyroid hormone 1-34 (nasal,osteoporosis), F-18-CCR1, AT-1001 (celiac disease/diabetes), JPD-003,PTH(7-34) liposomal cream (Novasome), duramycin (ophthalmic, dry eye),CAB-2, CTCE-0214, GlycoPEGylated erythropoietin, EPO-Fc, CNTO-528,AMG-114, JR-013, Factor XIII, aminocandin, PN-951, 716155, SUN-E7001,TH-0318, BAY-73-7977, teverelix (immediate release), EP-51216, hGH(controlled release, Biosphere), OGP-I, sifuvirtide, TV-4710, ALG-889,Org-41259, rhCCIO, F-991, thymopentin (pulmonary diseases), r(m)CRP,hepatoselective insulin, subalin, L 19-IL-2 fusion protein, elafin,NMK-150, ALTU-139, EN-122004, rhTPO, thrombopoietin receptor agonist(thrombocytopenic disorders), AL-108, AL-208, nerve growth factorantagonists (pain), SLV-317, CGX-1007, INNO-105, oral teriparatide(eligen), GEM-OS1, AC-162352, PRX-302, LFn-p24 fusion vaccine(Therapore), EP-1043, S. pneumoniae pediatric vaccine, malaria vaccine,Neisseria meningitidis Group B vaccine, neonatal group B streptococcalvaccine, anthrax vaccine, HCV vaccine (gpE1+gpE2+MF-59), otitis mediatherapy, HCV vaccine (core antigen+ISCOMATRIX), hPTH(1-34) (transdermal,ViaDerm), 768974, SYN-101, PGN-0052, aviscumine, BIM-23190, tuberculosisvaccine, multi-epitope tyrosinase peptide, cancer vaccine, enkastim,APC-8024, G1-5005, ACC-OO1, TTS-CD3, vascular-targeted TNF (solidtumors), desmopressin (buccal controlled-release), onercept, TP-9201.

Additional Modifications

In certain embodiments, the serum albumin binders and their fusions mayfurther comprise post-translational modifications. Exemplarypost-translational protein modifications include phosphorylation,acetylation, methylation, ADP-ribosylation, ubiquitination,glycosylation, carbonylation, sumoylation, biotinylation or addition ofa polypeptide side chain or of a hydrophobic group. As a result, themodified serum albumin binders and their fusions s may contain non-aminoacid elements, such as lipids, poly- or mono-saccharide, and phosphates.A preferred form of glycosylation is sialylation, which conjugates oneor more sialic acid moieties to the polypeptide. Sialic acid moietiesimprove solubility and serum half-life while also reducing the possibleimmunogenicity of the protein. See, e.g., Raju et al. Biochemistry. 2001Jul. 31; 40(30):8868-76. Effects of such non-amino acid elements on thefunctionality of the serum albumin binders or their fusions may betested for their ability to bind a particular serum albumin (e.g., HSAor RhSA) and/or the functional role conferred by a specific non-¹⁰Fn3moiety in the context of a fusion.

F. Nucleic Acid-Protein Fusion Technology

In one aspect, the application provides fibronectin based scaffoldproteins comprising a fibronectin type III domain that binds to HSA. Oneway to rapidly make and test Fn3 domains with specific bindingproperties is the nucleic acid-protein fusion technology of Adnexus, aBristol-Myers Squibb Company. Such in vitro expression and taggingtechnology, termed PROfusion, that exploits nucleic acid-protein fusions(RNA- and DNA-protein fusions) may be used to identify novelpolypeptides and amino acid motifs that are important for binding toproteins. Nucleic acid-protein fusion technology is a technology thatcovalently couples a protein to its encoding genetic information. For adetailed description of the RNA-protein fusion technology andfibronectin-based scaffold protein library screening methods see Szostaket al., U.S. Pat. Nos. 6,258,558; 6,261,804; 6,214,553; 6,281,344;6,207,446; 6,518,018; PCT Publication Nos. WO0/34784; WO01/64942;WO02/032925; and Roberts and Szostak, Proc Natl. Acad. Sci. 94:12297-12302, 1997, herein incorporated by reference.

G. Vectors & Polynucleotides Embodiments

Also included in the present disclosure are nucleic acid sequencesencoding any of the proteins described herein. As appreciated by thoseskilled in the art, because of third base degeneracy, almost every aminoacid can be represented by more than one triplet codon in a codingnucleotide sequence. In addition, minor base pair changes may result ina conservative substitution in the amino acid sequence encoded but arenot expected to substantially alter the biological activity of the geneproduct. Therefore, a nucleic acid sequence encoding a protein describedherein may be modified slightly in sequence and yet still encode itsrespective gene product. Certain exemplary nucleic acids encoding theserum albumin binders and their fusions described herein include nucleicacids having the sequences set forth in SEQ ID NOs: 126-151. Alsoencompassed by the invention are nucleic acid sequences that are atleast 50%, such as at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identicalto SEQ ID NOs: 126-151, and preferably encode a protein that binds toserum albumin, and for nucleic acids encoding a tandem PCSK9-PKE2Adnectin, that they preferably bind to serum albumin and PCSK9. In someembodiments, nucleotide substitutions are introduced so as not to alterthe resulting translated amino acid sequence.

Nucleic acids encoding any of the various proteins or polypeptidesdisclosed herein may be synthesized chemically. Codon usage may beselected so as to improve expression in a cell. Such codon usage willdepend on the cell type selected. Specialized codon usage patterns havebeen developed for E. coli and other bacteria, as well as mammaliancells, plant cells, yeast cells and insect cells. See for example:Mayfield et al., Proc Natl Acad Sci USA. 2003 100(2):438-42; Sinclair etal. Protein Expr Purif. 2002 (1):96-105; Connell ND. Curr OpinBiotechnol. 2001 (5):446-9; Makrides et al. Microbiol. Rev. 199660(3):512-38; and Sharp et al. Yeast. 1991 7(7):657-78.

General techniques for nucleic acid manipulation are within the purviewof one skilled in the art and are also described for example in Sambrooket al., Molecular Cloning: A Laboratory Manual, Vols. 1-3, Cold SpringHarbor Laboratory Press, 2 ed., 1989, or F. Ausubel et al., CurrentProtocols in Molecular Biology (Green Publishing and Wiley-Interscience:New York, 1987) and periodic updates, herein incorporated by reference.The DNA encoding a protein is operably linked to suitabletranscriptional or translational regulatory elements derived frommammalian, viral, or insect genes. Such regulatory elements include atranscriptional promoter, an optional operator sequence to controltranscription, a sequence encoding suitable mRNA ribosomal bindingsites, and sequences that control the termination of transcription andtranslation. The ability to replicate in a host, usually conferred by anorigin of replication, and a selection gene to facilitate recognition oftransformants are additionally incorporated. Suitable regulatoryelements are well-known in the art.

The proteins and fusion proteins described herein may be produced as afusion protein with a heterologous polypeptide, which is preferably asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. The heterologoussignal sequence selected preferably is one that is recognized andprocessed (i.e., cleaved by a signal peptidase) by the host cell. Forprokaryotic host cells that do not recognize and process a native signalsequence, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group of the alkalinephosphatase, penicillinase, 1 pp, or heat-stable enterotoxin II leaders.For yeast secretion, the native signal sequence may be substituted by,e.g., the yeast invertase leader, a factor leader (includingSaccharomyces and Kluyveromyces alpha-factor leaders), or acidphosphatase leader, the C. albicans glucoamylase leader, or the signaldescribed in PCT Publication No. WO 90/13646. In mammalian cellexpression, mammalian signal sequences as well as viral secretoryleaders, for example, the herpes simplex gD signal, are available. TheDNA for such precursor regions may be ligated in reading frame to DNAencoding the protein.

Expression vectors used in eukaryotic host cells (e.g., yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding the multivalent antibody.One useful transcription termination component is the bovine growthhormone polyadenylation region. See PCT Publication No. WO 94/11026 andthe expression vector disclosed therein.

The recombinant DNA can also include any type of protein tag sequencethat may be useful for purifying the protein. Examples of protein tagsinclude but are not limited to a histidine tag, a FLAG tag, a myc tag,an HA tag, or a GST tag. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts can befound in Cloning Vectors: A Laboratory Manual, (Elsevier, New York,1985), the relevant disclosure of which is hereby incorporated byreference.

The expression construct is introduced into the host cell using a methodappropriate to the host cell, as will be apparent to one of skill in theart. A variety of methods for introducing nucleic acids into host cellsare known in the art, including, but not limited to, electroporation;transfection employing calcium chloride, rubidium chloride, calciumphosphate, DEAE-dextran, or other substances; microprojectilebombardment; lipofection; and infection (where the vector is aninfectious agent).

Suitable host cells include prokaryotes, yeast, mammalian cells, orbacterial cells. Suitable bacteria include gram negative or grampositive organisms, for example, E. coli or Bacillus spp. Yeast,preferably from the Saccharomyces species, such as S. cerevisiae, mayalso be used for production of polypeptides. Various mammalian or insectcell culture systems can also be employed to express recombinantproteins. Baculovirus systems for production of heterologous proteins ininsect cells are reviewed by Luckow and Summers, (Bio/Technology, 6:47,1988). In some instance it will be desired to produce proteins invertebrate cells, such as for glycosylation, and the propagation ofvertebrate cells in culture (tissue culture) has become a routineprocedure. Examples of suitable mammalian host cell lines includeendothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3,Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293,293T, and BHK cell lines. For many applications, the small size of theprotein multimers described herein would make E. coli the preferredmethod for expression.

H. Protein Production

Host cells are transformed with the herein-described expression orcloning vectors for protein production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.The host cells used to produce the fibronectin based scaffold proteinsor fusions thereof may be cultured in a variety of media. Commerciallyavailable media such as Ham's F10 (Sigma), Minimal Essential Medium((MEM), (Sigma)), RμMI-1640 (Sigma), and Dulbecco's Modified Eagle'sMedium ((DMEM), (Sigma)) are suitable for culturing the host cells. Inaddition, any of the media described in Ham et al., Meth. Enz. 58:44(1979), Barnes et al., Anal. Biochem.102:255 (1980), U.S. Pat. Nos.4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90/03430;WO87/00195; or U.S. Pat. No. Re. 30,985 may be used as culture media forthe host cells. Any of these media may be supplemented as necessary withhormones and/or other growth factors (such as insulin, transferrin, orepidermal growth factor), salts (such as sodium chloride, calcium,magnesium, and phosphate), buffers (such as HEPES), nucleotides (such asadenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), traceelements (defined as inorganic compounds usually present at finalconcentrations in the micromolar range), and glucose or an equivalentenergy source. Any other necessary supplements may also be included atappropriate concentrations that would be known to those skilled in theart. The culture conditions, such as temperature, pH, and the like, arethose previously used with the host cell selected for expression, andwill be apparent to the ordinarily skilled artisan.

Fibronectin based scaffold proteins disclosed herein or fusions thereofcan also be produced using cell-free translation systems. For suchpurposes the nucleic acids encoding the fibronectin based scaffoldprotein must be modified to allow in vitro transcription to produce mRNAand to allow cell-free translation of the mRNA in the particularcell-free system being utilized (eukaryotic such as a mammalian or yeastcell-free translation system or prokaryotic such as a bacterialcell-free translation system).

Fibronectin based scaffold proteins or fusions thereof can also beproduced by chemical synthesis (e.g., by the methods described in SolidPhase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co.,Rockford, Ill.). Modifications to the fibronectin based scaffold proteincan also be produced by chemical synthesis.

The fibronectin based scaffold proteins disclosed herein or fusionsthereof can be purified by isolation/purification methods for proteinsgenerally known in the field of protein chemistry. Non-limiting examplesinclude extraction, recrystallization, salting out (e.g., with ammoniumsulfate or sodium sulfate), centrifugation, dialysis, ultrafiltration,adsorption chromatography, ion exchange chromatography, hydrophobicchromatography, normal phase chromatography, reversed-phasechromatography, gel filtration, gel permeation chromatography, affinitychromatography, electrophoresis, countercurrent distribution or anycombinations of these. After purification, fibronectin based scaffoldproteins may be exchanged into different buffers and/or concentrated byany of a variety of methods known to the art, including, but not limitedto, filtration and dialysis.

The purified fibronectin based scaffold protein or fusions thereof ispreferably at least 85% pure, more preferably at least 95% pure, andmost preferably at least 98% pure. Regardless of the exact numericalvalue of the purity, the fibronectin based scaffold protein issufficiently pure for use as a pharmaceutical product.

I. Imaging, Diagnostic, and Other Applications

The serum albumin binding ¹⁰Fn3 fusions provided herein may be used totreat a variety of diseases and disorders, based on the identity of theheterogenous molecule fused to the serum albumin binding ¹⁰Fn3 domain.The applications for the serum albumin binding ¹⁰Fn3 fusions may bedetermined by the skilled artisan based on the knowledge in the art andthe information provided herein. Uses for various serum albumin binding¹⁰Fn3 fusion proteins are described in detail herein. Serum albuminbinding ¹⁰Fn3fusions may be administered to any mammalian subject orpatient, including both human and non-human organisms.

The serum albumin binders and fusion molecules described herein can bedetectably labeled and used to contact cells expressing, e.g., a proteinbound by the fusion molecule for imaging or diagnostic applications. Anymethod known in the art for conjugating a protein to the detectablemoiety may be employed, including those methods described by Hunter, etal., Nature 144:945 (1962); David, et al., Biochemistry 13:1014 (1974);Pain, et al., J. Immunol. Meth. 40:219 (1981); and Nygren, J. Histochem.and Cytochem. 30:407 (1982).

In certain embodiments, the serum albumin binders and fusion moleculesdescribed herein are further attached to a label that is able to bedetected (e.g., the label can be a radioisotope, fluorescent compound,enzyme or enzyme co-factor). The label may be a radioactive agent, suchas: radioactive heavy metals such as iron chelates, radioactive chelatesof gadolinium or manganese, positron emitters of oxygen, nitrogen, iron,carbon, or gallium, ⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, 68Ga, ¹²³I, ¹²⁵I ¹³¹I,¹³²I, or ⁹Tc. In certain embodiments, the label can be a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. A serum albumin binder orfusion molecule affixed to such a moiety may be used as an imaging agentand is administered in an amount effective for diagnostic use in amammal such as a human and the localization and accumulation of theimaging agent is then detected. The localization and accumulation of theimaging agent may be detected by radioscintigraphy, nuclear magneticresonance imaging, computed tomography or positron emission tomography.As will be evident to the skilled artisan, the amount of radioisotope tobe administered is dependent upon the radioisotope. Those havingordinary skill in the art can readily formulate the amount of theimaging agent to be administered based upon the specific activity andenergy of a given radionuclide used as the active moiety.

Serum albumin binders and fusion molecules also are useful as affinitypurification agents. In this process, the proteins are immobilized on asuitable support, such a Sephadex resin or filter paper, using methodswell known in the art. The proteins can be employed in any known assaymethod, such as competitive binding assays, direct and indirect sandwichassays, and immunoprecipitation assays (Zola, Monoclonal Antibodies: AManual of Techniques, pp. 147-158 (CRC Press, Inc., 1987)).

J. Biophysical and Biochemical Characterization

Binding of a serum albumin binding Adnectin described herein to serumalbumin (e.g., HSA) may be assessed in terms of equilibrium constants(e.g., dissociation, K_(D)) and in terms of kinetic constants (e.g.,on-rate constant, k_(on) and off-rate constant, k_(off)). A serumalbumin binding Adnectin (e.g., a PKE2-mono- or tandem-Adenctin) willgenerally bind to a target molecule with a K_(D) of less than 500 nM,100 nM, 10 nM, 1 nM, 500 μM, 200 μM, or 100 μM, although higher K_(D)values may be tolerated where the k_(off) is sufficiently low or thek_(on), is sufficiently high.

In Vitro Assays for Binding Affinity

A PKE2-Adnectin that binds to serum albumin (e.g., HSA) can beidentified using various in vitro assays. In certain embodiments, theassays are high-throughput assays that allow for screening multiplecandidate Adnectins simultaneously.

Exemplary assays for determining the binding affinity of an Adnectin toits target includes, but is not limited to, solution phase methods suchas the kinetic exclusion assay (KinExA) (Blake et al., JBC 1996;271:27677-85; Drake et al., Anal Biochem 2004; 328:35-43), surfaceplasmon resonance (SPR) with the Biacore system (Uppsala, Sweden)(Welford et al. Opt. Quant. Elect 1991; 23:1; Morton and Myszka, Methodsin Enzymology 1998; 295:268) and homogeneous time resolved fluorescence(HTRF) assays (Newton et al., J Biomol Screen 2008; 13:674-82; Patel etal., Assay Drug Dev Technol 2008; 6:55-68).

In certain embodiments, biomolecular interactions can be monitored inreal time with the Biacore system, which uses SPR to detect changes inthe resonance angle of light at the surface of a thin gold film on aglass support due to changes in the refractive index of the surface upto 300 nm away. Biacore analysis (e.g., as described in Example 2)generates association rate constants, dissociation rate constants,equilibrium dissociation constants, and affinity constants. Bindingaffinity is obtained by assessing the association and dissociation rateconstants using a Biacore surface plasmon resonance system (Biacore,Inc.). A biosensor chip is activated for covalent coupling of thetarget. The target is then diluted and injected over the chip to obtaina signal in response units of immobilized material. Since the signal inresonance units (RU) is proportional to the mass of immobilizedmaterial, this represents a range of immobilized target densities on thematrix. Association and dissociation data are fit simultaneously in aglobal analysis to solve the net rate expression for a 1:1 bimolecularinteraction, yielding best fit values for k_(on), k_(off) and R_(max)(maximal response at saturation). Equilibrium dissociation constants forbinding, K_(D)'s are calculated from SPR measurements as k_(off)/k_(on).

It should be understood that the assays described herein above areexemplary, and that any method known in the art for determining thebinding affinity between proteins (e.g., fluorescence based-transfer(FRET), enzyme-linked immunosorbent assay, and competitive bindingassays (e.g., radioimmunoassays)) can be used to assess the bindingaffinities of the PKE2-Adnectins described herein.

In certain embodiments, the melting temperature (T_(m)) of serum albuminbinding Adnectin described herein, of fusion proteins comprising such,is at least 50° C., such as at least 51° C., at least 52° C. at least53° C. at least 54° C., at least 55° C., at least 56° C., at least 57°C., at least 58° C., at least 59° C., at least 60° C., at least 61° C.,at least 62° C., at least 63° C., at least 64° C., at least 65° C., atleast 66° C., at least 67° C. at least 68° C., at least 69° C., at least70° C., at least 71° C. at least 72° C., at least 73° C., at least 74°C., or at least 75° C., when measured using differential scanningcalorimetry (DSC) or thermal scanning fluorescence (TSF), e.g., asdescribed in the Examples. In certain embodiments, the meltingtemperature (T_(m)) of serum albumin binding Adnectin described herein,or fusion proteins comprising such, is 50-75° C., such as 51-75° C.,52-75° C., 53-75° C., 54-75° C., 55-75° C., 56-75° C., 57-75° C. 58-75°C., 59-75° C., 60-75° C., 61- 75° C., 62-75° C., 63-75° C., 64-75° C.,65-75° C., 66-75° C., 67-75° C., 68-75° C., 69-75° C., 70-75° C., 50-74° C., 50-73° C., 50-72° C. 50-71° C., 50-70° C., 50-69° C. 50-68° C.,50-67° C., 50-66° C., 50-65° C., 50-64° C. 50-63° C., 50-62° C., 50-61°C., 50-60° C., 50-59° C., 50-58° C., 50-57° C., 50-56° C., 50-55° C.,51- 74° C., 52-73° C., 53-71° C., 54-70° C., or 55-65° C. when measuredusing differential scanning calorimetry (DSC) or thermal scanningfluorescence (TSF), e.g., as described in the Examples.

K. Therapeutic In Vivo Uses

Provided herein are fibronectin based scaffold proteins that are usefulin the treatment of disorders. In the case of fusion proteins comprisinga serum albumin binding Adnectin, the diseases or disorders that may betreated will be dictated by the binding specificity of the moiety, e.g.,a second Adnectin, that is linked to the Adnectin. As described herein,fibronectin based scaffold proteins may be designed to bind to anytarget of interest. In one embodiment, the target is PCSK9. Fibronectinbased scaffold proteins that bind to PSCK9 and the fusion proteinscomprising such can be used to treat atherosclerosis,hypercholesterolemia and other cholesterol related diseases.

The application also provides methods for administering fibronectinbased scaffold proteins to a subject. In some embodiments, the subjectis a human. In some embodiments, the fibronectin based scaffold proteinsare pharmaceutically acceptable to a mammal, in particular a human. A“pharmaceutically acceptable” composition refers to a composition thatis administered to an animal without significant adverse medicalconsequences. Examples of pharmaceutically acceptable compositionsinclude compositions comprising ¹⁰Fn3 domains that lack theintegrin-binding domain (RGD) and compositions that are essentiallyendotoxin or pyrogen free or have very low endotoxin or pyrogen levels.

L. Formulations and Administration

The present application provides methods for administering a therapeuticmoiety fused to a serum albumin binding ¹⁰Fn3 domain, wherein thehalf-life of the therapeutic moiety is extended when fused to the serumalbumin binding ¹⁰Fn3 domain. Techniques and dosages for administrationof the fusion constructs will vary depending on the type of therapeuticmoiety fused to the serum albumin binding ¹⁰Fn3 domain and the specificcondition being treated but can be readily determined by the skilledartisan. In general, regulatory agencies require that a protein reagentto be used as a therapeutic is formulated so as to have acceptably lowlevels of pyrogens. Accordingly, therapeutic formulations will generallybe distinguished from other formulations in that they are substantiallypyrogen free, or at least contain no more than acceptable levels ofpyrogen as determined by the appropriate regulatory agency (e.g., FDA).In certain embodiments, pharmaceutical formulations of serum albuminbinding ¹⁰Fn3 domains and their fusion molecules comprise, e.g., 1-20 mMsuccinic acid, 2-10% sorbitol, and 1-10% glycine at pH 4.0-7.0. In anexemplary embodiment, pharmaceutical formulations of serum albuminbinding ¹⁰Fn3 domains and their fusion molecules comprise, e.g., 10 mMsuccinic acid, 8% sorbitol, and 5% glycine at pH 6.0.

In some embodiments, the serum albumin binding ¹⁰Fn3 domains and fusionsthereof are pharmaceutically acceptable to a mammal, in particular ahuman. A “pharmaceutically acceptable” polypeptide refers to apolypeptide that is administered to an animal without significantadverse medical consequences. Examples of pharmaceutically acceptableserum albumin binding ¹⁰Fn3 domain and fusions thereof include ¹⁰Fn3domains that lack the integrin-binding domain (RGD) and compositions ofserum albumin binding ¹⁰Fn3 domains or serum albumin binding ¹⁰Fn3domain fusions that are essentially endotoxin free or have very lowendotoxin levels.

Therapeutic compositions may be administered with a pharmaceuticallyacceptable diluent, carrier, or excipient, in unit dosage form.Administration may be parenteral (e.g., intravenous, subcutaneous),oral, or topical, as non-limiting examples. The composition can be inthe form of a pill, tablet, capsule, liquid, or sustained release tabletfor oral administration; a liquid for intravenous, subcutaneous orparenteral administration; or a gel, lotion, ointment, cream, or apolymer or other sustained release vehicle for local administration.

Methods well known in the art for making formulations are found, forexample, in “Remington: The Science and Practice of Pharmacy” (20th ed.,ed. A. R. Gennaro A R., 2000, Lippincott Williams & Wilkins,Philadelphia, Pa.). Formulations for parenteral administration may, forexample, contain excipients, sterile water, saline, polyalkylene glycolssuch as polyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds.Nanoparticulate formulations (e.g., biodegradable nanoparticles, solidlipid nanoparticles, liposomes) may be used to control thebiodistribution of the compounds. Other potentially useful parenteraldelivery systems include ethylene-vinyl acetate copolymer particles,osmotic pumps, implantable infusion systems, and liposomes. Theconcentration of the compound in the formulation varies depending upon anumber of factors, including the dosage of the drug to be administered,and the route of administration.

The polypeptide may be optionally administered as a pharmaceuticallyacceptable salt, such as non-toxic acid addition salts or metalcomplexes that are commonly used in the pharmaceutical industry.Examples of acid addition salts include organic acids such as acetic,lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic,palmitic, suberic, salicylic, tartaric, methanesulfonic,toluenesulfonic, or trifluoroacetic acids or the like; polymeric acidssuch as tannic acid, carboxymethyl cellulose, or the like; and inorganicacid such as hydrochloric acid, hydrobromic acid, sulfuric acidphosphoric acid, or the like. Metal complexes include zinc, iron, andthe like. In one example, the polypeptide is formulated in the presenceof sodium acetate to increase thermal stability.

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose and sorbitol), lubricating agents, glidants, andanti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid,silicas, hydrogenated vegetable oils, or talc).

Formulations for oral use may also be provided as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent, or as soft gelatin capsules wherein the activeingredient is mixed with water or an oil medium.

A therapeutically effective dose refers to a dose that produces thetherapeutic effects for which it is administered. The exact dose willdepend on the disorder to be treated, and may be ascertained by oneskilled in the art using known techniques. In general, the serum albuminbinding ¹⁰Fn3 domains or serum albumin binding ¹⁰Fn3 domain fusion isadministered at about 0.01 μg/kg to about 50 mg/kg per day, preferably0.01 mg/kg to about 30 mg/kg per day, most preferably 0.1 mg/kg to about20 mg/kg per day. The polypeptide may be given daily (e.g., once, twice,three times, or four times daily) or less frequently (e.g., once everyother day, once or twice weekly, once every two weeks, or monthly). Inaddition, as is known in the art, adjustments for age as well as thebody weight, general health, sex, diet, time of administration, druginteraction, and the severity of the disease may be necessary, and willbe ascertainable with routine experimentation by those skilled in theart.

The contents of all figures and all references, Genbank sequences,patents and published patent applications cited throughout thisapplication are expressly incorporated herein by reference. Inparticular, the disclosure of U.S. Provisional Patent Application No.61/968,181 (filed on Mar. 20, 2014) is expressly incorporated herein byreference.

The above disclosure generally describes the present disclosure, whichis further exemplified by the following examples. These specificexamples are described solely for purposes of illustration, and are notintended to limit the scope of this disclosure. Although specifictargets, terms, and values have been employed herein, such targets,terms, and values will likewise be understood as exemplary andnon-limiting to the scope of this disclosure.

EXAMPLES High Throughput Protein Production (HTPP)

Selected binders cloned into the PET9d vector upstream of a HIS₆tag andtransformed into E. coli BL21 DE3 plysS cells were inoculated in 5 ml LBmedium containing 50 g±g/mL kanamycin in a 24-well format and grown at37° C. overnight. Fresh 5 ml LB medium (50 μg/mL kanamycin) cultureswere prepared for inducible expression by aspiration of 200 μl from theovernight culture and dispensing it into the appropriate well. Thecultures were grown at 37° C. until A₆₀₀ 0.6-0.9. After induction with 1mM isopropyl-p-thiogalactoside (IPTG), the culture was expressed for 6hours at 30° C. and harvested by centrifugation for 10 minutes at 2750 gat 4° C.

Cell pellets (in 24-well format) were lysed by resuspension in 450 μl ofLysis buffer (50 mM NaH₂PO₄, 0.5 M NaCl, 1× Complete m ProteaseInhibitor Cocktail-EDTA free (Roche), 1 mM μMSF, 10 mM CHAPS, 40 mMimidazole, 1 mg/ml lysozyme, 30 g±g/ml DNAse, 2 μg/ml aprotonin, pH 8.0)and shaken at room temperature for 1-3 hours. Lysates were cleared andre-racked into a 96-well format by transfer into a 96-well Whatman GF/DUnifilter fitted with a 96-well, 1.2 ml catch plate and filtered bypositive pressure. The cleared lysates were transferred to a 96-wellNickel or Cobalt-Chelating Plate that had been equilibrated withequilibration buffer (50 mM NaH₂PO₄, 0.5 M NaCl, 40 mM imidazole, pH8.0) and were incubated for 5 min. Unbound material was removed bypositive pressure. The resin was washed twice with 0.3 ml/well with Washbuffer #1 (50 mM NaH₂PO₄, 0.5 M NaCl, 5 mM CHAPS, 40 mM imidazole, pH8.0). Each wash was removed by positive pressure. Prior to elution, eachwell was washed with 50 μl Elution buffer (PBS+20 mM EDTA), incubatedfor 5 min, and this wash was discarded by positive pressure. Protein waseluted by applying an additional 100 μl of Elution buffer to each well.After a 30 minute incubation at room temperature, the plate(s) werecentrifuged for 5 minutes at 200 g and eluted protein collected in96-well catch plates containing 5 μl of 0.5 M MgCl₂ added to the bottomof elution catch plate prior to elution. Eluted protein was quantifiedusing a total protein assay with wild-type ¹⁰Fn3 domain as the proteinstandard.

Midscale Expression and Purification of Insoluble Fibronectin-BasedScaffold Protein Binders

For expression of insoluble clones, the clone(s), followed by theHIS₆tag, are cloned into a pET9d (EMD Bioscience, San Diego, Calif.)vector and are expressed in E. coli HMS174 cells. Twenty ml of aninoculum culture (generated from a single plated colony) is used toinoculate 1 liter of LB medium containing 50 μg/ml carbenicillin and 34μg/ml chloramphenicol. The culture is grown at 37° C. until A₆₀₀0.6-1.0. After induction with 1 mM isopropyl-β-thiogalactoside (IPTG)the culture is grown for 4 hours at 30° C. and is harvested bycentrifugation for 30 minutes at >10,000 g at 4° C. Cell pellets arefrozen at −80° C. The cell pellet is resuspended in 25 ml of lysisbuffer (20 mM aH2P0₄, 0.5 M NaCl, lx Complete Protease InhibitorCocktail-EDTA free (Roche), ImM μMSF, pH 7.4) using an ULTRA-TURRAX®homogenizer (IKA works) on ice. Cell lysis is achieved by high pressurehomongenization (>18,000 psi) using a Model M-1 10S MICROFLUIDIZER®(Microfluidics). The insoluble fraction is separated by centrifugationfor 30 minutes at 23,300 g at 4° C. The insoluble pellet recovered fromcentrifugation of the lysate is washed with 20 mM sodiumphosphate/500 mMNaCl, pH7.4. The pellet is resolubilized in 6.0M guanidine hydrochloridein 20 mM sodium phosphate/500M NaCl pH 7.4 with sonication followed byincubation at 37 degrees for 1-2 hours. The resolubilized pellet isfiltered to 0.45 μm and loaded onto a Histrap column equilibrated withthe 20 mM sodium phosphate/500 M NaCl/6.0 M guanidine pH 7.4 buffer.After loading, the column is washed for an additional 25 CV with thesame buffer. Bound protein is eluted with 50 mM Imidazole in 20 mMsodium phosphate/500 mM NaCl/6.0 M guan-HCl pH7.4. The purified proteinis refolded by dialysis against 50 mM sodium acetate/150 mM NaCl pH 4.5.

Midscale Expression and Purification of Soluble Fibronectin-BaseScaffold Protein Binders

For expression of soluble clones, the clone(s), followed by the HIS₆tag,were cloned into a pET9d (EMD Bioscience, San Diego, Calif.) vector andwere expressed in E. coli HMS174 cells. Twenty ml of an inoculum culture(generated from a single plated colony) was used to inoculate 1 liter ofLB medium containing 50 μg/ml carbenicillin and 34 μg/mlchloramphenicol. The culture was grown at 37° C. until A₆₀₀ 0.6-1.0.After induction with 1 mM isopropyl-3-thiogalactoside (IPTG), theculture was grown for 4 hours at 30° C. and was harvested bycentrifugation for 30 minutes at >10,000 g at 4° C. Cell pellets werefrozen at −80° C. The cell pellet was resuspended in 25 ml of lysisbuffer (20 mM NaH₂P0₄, 0.5 M NaCl, lx Complete Protease InhibitorCocktail-EDTA free (Roche), ImM μMSF, pH 7.4) using an ULTRA-TURRAX®homogenizer (IKA works) on ice. Cell lysis was achieved by high pressurehomongenization (>18,000 psi) using a Model M-1 10S MICROFLUIDIZER®(Microfluidics). The soluble fraction was separated by centrifugationfor 30 minutes at 23,300 g at 4° C. The supernatant was clarified via0.45 μm filter. The clarified lysate was loaded onto a Histrap column(GE) pre-equilibrated with the 20 mM sodium phosphate/500M NaCl pH 7.4.The column was then washed with 25 column volumes of the same buffer,followed by 20 column volumes of 20 mM sodium phosphate/500 M NaCl/25 mMImidazole, pH 7.4 and then 35 column volumes of 20 mM sodiumphosphate/500 M NaCl/40 mM Imidazole, pH 7.4. Protein was eluted with 15column volumes of 20 mM sodium phosphate/500 M NaCl/500 mM Imidazole, pH7.4, fractions were pooled based on absorbance at A₂₈₀ and were dialyzedagainst 1×PBS, 50 mM Tris, 150 mM NaCl; pH 8.5 or 50 mM NaOAc; 150 mMNaCl; pH4.5. Any precipitate was removed by filtering at 0.22 μm.

Example 1: Screening for Serum Albumin Binding Parent South Loop (CDLoop)-Based Binders

In order to improve upon first generation north pole-based serum albuminbinding Adnectins (SABAs) which did not bind to mouse and rat serumalbumin, did not have high affinity for serum albumins across species,and were not always compatible in a multivalent ¹⁰Fn3-based platform,second generation south pole-based serum albumin binding Adnectins (PKE2Adnectins) with modified CD loop sequences were screened using mRNAdisplay as described below.

Libraries of CD loop-based binder polypeptides comprising a modified¹⁰Fn3 domain were screened using mRNA display (Xu et al., Chem Biol2002; 9:933-42) for the ability to bind to human serum albumin (HSA).The CD loop binders were designed with varying CD loop lengths up to +7amino acids and the rest of the ¹⁰Fn3 sequence remained wildtype. Targetbinding was monitored by qPCR and populations were cloned and expressedin E. Coli when a specific binding signal was observed.

Example 2: Identification of CD Loop Binders Capable of Binding HSA andthat Cross-React with Rh-SA and MSA

A direct binding ELISA format was used to identify CD loop binders thatwere generated in Example 1 and that bound HSA and cross-reacted withrhesus serum albumin (Rh-SA) and/or with murine serum albumin (MSA).MaxiSorp™ ELISA plates were coated with 10 μg/mL of either HSA, Rh-SA,or MSA and purified CD loop binders were tested at 1 μM. Bound Adnectinswere detected via an HRP-conjugated anti-histidine mAb (R&D Systems) andthe TMB detection reagents (BD Biosciences). The ELISA results wereconfirmed using Biacore as described below. CD loop binders identifiedin the ELISA experiment as cross-reacting with Rh-SA and/or MSA (>2×background) were then analyzed by SEC for aggregation in order todemonstrate that binding was due to a monomeric species, as expected ofa stable, well-folded protein. The stability of the protein wasconfirmed by differential scanning calorimetry (DSC) as described below.

One of the identified clones, herein referred to as 2270_C01, had thefollowing amino acid sequence:

(2270_C01; SEQ ID NO: 23)MASTSGVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGWQVQMYSDWGPLYIYKEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPAS SKPISINYRTEGDKPSQHHHHHH

The CD loop is underlined. The AB, BC, DE, EF, and FG loops havesequences identical to the wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1).Size exclusion chromatography and DSC analyses were performed onmidscaled 2270_C01 to confirm monomericity and determine thermalstability.

Standard size exclusion chromatography (SEC) was performed on 2270_C01resulting from the midscale process. SEC of midscaled material wasperformed using a Superdex 200 10/30 or on a Superdex 75 10/30 column(GE Healthcare) on an Agilent 1100 or 1200 HPLC system with UV detectionat A214 nm and A280 nm and with fluorescence detection (excitation=280nm, emission=350 nm). A buffer of 100 mM sodium sulfate, 100 mM sodiumphosphate, 150 mM sodium chloride, pH 6.8 at an appropriate flow rate ofthe SEC column was employed. Gel filtration standards (Bio-RadLaboratories, Hercules, Calif.) were used for molecular weightcalibration. As shown in Table 2, 2270_C01 was primarily monomeric (98%monomer).

Differential Scanning Calorimetry (DSC) analyses of the midscaledAdnectins were performed to determine their respective T_(m)'s. A 0.5mg/ml solution was scanned in a VP-Capillary Differential Scanningcalorimeter (GE Microcal) by ramping the temperature from 15° C. to 110°C. at a rate of 1 degree per minute under 70 p.s.i pressure. The datawas analyzed vs. a control run of the appropriate buffer using a bestfit using Origin Software (OriginLab Corp). As shown in Table 2,2270_C01 had a Tm of 64° C.

To determine the kinetics of binding to human, rhesus, and mouse serumalbumin, as well as whether binding was retained at both physiologicaland endosomal pHs, the respective serum albumins were immobilized on aBiacore CM5 chip to a surface density of ˜1200RU using standard NHS/EDCcoupling. A concentration range (0.25 nM-5 uM) of 2270_C01 was appliedin HBS-P+(0.01M HEPES pH 7.4, 0.15M NaCl, 0.05% v/v surfactant P-20) orAcetate (0.02 mM sodium acetate pH 5.5, 0.15M NaCl, 0.05% v/v surfactantP-20) running buffers to the immobilized albumins. Kinetic measurementswere carried out using a 3 min association and 6-10 min dissociationphase. Kinetic traces of reference-subtracted sensorgrams were fit to a1:1 binding model using the Biaevaluation software. As shown in Table 1,2270_C01 bound with equivalent affinity at neutral and low pH to eachspecies of albumin, however the affinity for mouse albumin wasapproximately 10-fold weaker than that of binding to human or rhesusalbumin.

TABLE 1 2270_C01 binds to MuSA with slightly faster on-rates andsignificantly faster off-rates, compared to HuSA and RhSA at both pH 7.4and 5.5 Rmax Buffer Binding to ka (1/Ms) kd (1/s) KD (nM) (RU) HBS-P, pH7.4 HuSA 6.59E+04 3.68E−04 5.58 121.6 RhSA 8.27E+04 5.77E−04 6.98 103.3MuSA 1.34E+05 9.09E−03 67.67 77.64 Acetate, pH 5.5 HuSA 1.02E+058.98E−04 8.82 111.9 RhSA 5.96E+04 1.05E−03 17.55 85.5 MuSA 7.59E+041.46E−02 ~192.4 57.91

To improve on properties of 2270_C01, namely in silico predictedimmunogenicity, the 2270_C01 sequence was subjected to optimization bymRNA display. Resulting Adnectins from this optimization are hereinreferred to as PKE2 Adnectins.

Example 3: Generation of 2270_C01 Progeny Adnectins with FurtherModified CD Loop: PKE2 Adnectins

The 2270_C01 sequence was subjected to optimization by mRNA displayutilizing custom-designed libraries to reduce immunogenicity potential,and screened for binding to both human and mouse serum albumins duringthe mRNA display process in order to obtain lower immunogenicity progenymolecules that retained cross-species albumin binding. Resultingsequences were evaluated for in silico predicted immunogenicity and onlyclones that had an in silico immunogenicity score lower than thepre-determined cut-off were progressed into protein production by HTPP.Resulting Adnectins were purified by HTPP and screened by direct bindingELISA and SEC-HPLC as described above.

Of the 308 PKE2 Adnectins obtained in the screening of 2270_C01 progenyand tested, the following 25 were the best performing molecules in termsof in silico predicted immunogenicity, monomericity as determined by SECand binding to serum albumin from various species as determined bydirect binding ELISA. Affinity determinations of the top candidates wereanalyzed by SPR as described above.

SEQ PKE2 Sequence ID Adnectins (with CD loop underlined) 24 2629_A09MGVSDVPRDLEVVAATPTSLLISWDAPA VTVRYYRITYGRHVQIYSDLGPLYIYTEFTVPGSKSTATISGLKPGVDYTITVYAV TGSGESPASSKPISINYRTEIDKPSQHH HHHH 252629_A11 MGVSDVPRDLEVVAATPTSLLISWDAPA VTVRYYRITYGRHVHIYSDWGPMYIYTEFTVPGSKSTATISGLKPGVDYTITVYAV TGSGESPASSKPISINYRTEIDKPSQHH HHHH 262629_C10 MGVSDVPRDLEVVAATPTSLLISWDAPA VTVRYYRITYGREVQKYSVLGPLYIYTEFTVPGSKSTATISGLKPGVDYTITVYAV TGSGESPASSKPISINYRTEIDKPSQHH HHHH 272629_D09 MGVSDVPRDLEVVAATPTSLLISWDAPA VTVRYYRITYGREVQMYSDLGPLYVYSEFTVPGSKSTATISGLKPGVDYTITVYAV TGSGESPASSKPISINYRTEIDKPSQHH HHHH 282629_E05 MGVSDVPRDLEVVAATPTSLLISWDAPA VTVRYYRITYGREVQKFSDWGPLYIYTEFTVPGSKSTATISGLKPGVDYTITVYAV TGSGESPASSKPISINYRTEIDKPSQHH HHHH 292629_E06 MGVSDVPRDLEVVAATPTSLLISWDAPA VTVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAV TGSGESPASSKPISINYRTEIDKPSQHH HHHH 302629_F04 MGVSDVPRDLEVVAATPTSLLISWDAPA VTVRYYRITYGREVHQYSDWGPMYIYNEFTVPGSKSTATISGLKPGVDYTITVYAV TGSGESPASSKPISINYRTEIDKPSQHH HHHH 312629_H01 MGVSDVPRDLEVVAATPTSLLISWDAPA VTVXYYRITYGREVHKNSDWGTLYIYTEFTVPGSKSTATISGLKPGVDYTITVXAV TGSGEXPASSKPISINYRTEIDKXSQHH HHHH 322629_H06 MGVSDVPRDLEVVAATPTSLLISWDAPA VTVRYYRITYGREVQKYSDLGPLYIYAEFTVPGSKSTATISGLKPGVDYTITVYAV TGSGESPASSKPISINYRTEIDKPSQHH HHHH 332629_H07 MGVSDVPRDLEVVAATPTSLLISWDAPA VTVRYYRITYGREVHLYSDWGPMYIYTEFTVPGSKSTATISGLKPGVDYTITVYAV TGSGESPASSKPISINYRTEIDKPSQHH HHHH 342630_A02 MGVSDVPRDLEVVATTPTSLLISWDAPA VTVRYYRITYGRHVQMYSDLGPLYIFSEFTVPGSKSTATISGLKPGVDYTITVYAV TGSGESPASSKPISINYRTEIDKPSQHH HHHH 352630_A11 MGVSDVPRDLEVVAATPTSLLISWDAPA VTVRYYRITYGREVHMYSDFGPMYIYTEFTVPGSKSTATISGLKPGVDYTITVYAV TGSGESPASSKPISINYRTEIDKPSQHH HHHH 362630_D02 MGVSDVPRDLEVVAATPTSLLISWDAPA VTVRYYRITYGREVQKYSDWGPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAV TGSGESPASSKPISINYRTEIDKPSQHH HHHH 372630_D10 MGVSDVPRDLEVVAATPTSLLISWDAPA VTVRYYRITYGREVQMYSDLGPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAV TGSGESPASSKPISINYRTEIDKPSQHH HHHH 382630_F04 MGVSDVPRDLEVVAATPTSLLISWDAPA VTVRYYRITYGREVQMYSDLGPLYIYTEFTVPGSKSTATISGLKPGVGYTITVYAV TGSGESPASSKPISINYRTEIDKPSQHH HHHH 392630_G03 MGVSDVPRDLEVVAATPTSLLISWDAPA VTVRYYRITYGRHVQIYSDLGPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAV TGSGESPASSKPISINYRTEIDKPSQHH HHHH 402630_G10 MGVSDVPRDLEVVAATPTSLLISWDAPA VTVRYYRITYGREVQIYSDWGPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAV TGSGESPASSKPISINYRTEIDKPSQHH HHHH 412630_H03 MGVSDVPRDLEVVAATXTSLLISWDAPA VTVXYYRITYGREVQKYSDWGPLYIYQEFTVPGSXSTATISGLKPGVDYTITVYAV TGSGESPASSKPISINYRTEIDKXSQHH HHHH 422631_B04 MGVSDVPRDLEVVAATPTSLLISWDVPA VTVRYYRITYGRHVHLYSEFGPMYIYNEFTVPGSKSTATISGLKPGVDYTITVYAV TGSGESPASSKPISINYRTEIDKPSQHH HHHH 432631_E03 MGVSDVPRDLEVVAATPTSLLISWDAPA VTVRYYRITYGRDVHMYSDWGPMYIYQEFTVPGSKSTATISGLKPGVDYTITVYAV TGSGESPASSKPISINYRTEIDKPSQHH HHHH 442631_G01 MGVSDVPRDLEVVAATPTSLLISWDAPA VTVRYYRITYGRHVQIYSDWGPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAV TGSGESPASSKPISINYRTEIDKPSQHH HHHH 452631_G03 MGVSDVPRDLEVVAATPTSLLISWDAPA VTVRYYRITYGRYVQLYSDWGPMYIYTEFTVPGSKSTATISGLKPGVDYTITVYAV TGSGESPASSKPISINYRTEIDKPSQHH HHHH 462631_H09 MGVSDVPRDLEVVAATPTSLLISWDAPA VTVRYYRITYGRQVQVFSDLGPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAV TGSGESPASSKPISINYRTEIDKPSQHH HHHH 472632_G01 MGVSDVPRDLEVVAATPTSLLISWDAPA VTVRYYRITYGRQVQIYSDWGPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAV TGSGESPASSKPISINYRTEIDKPSQHH HHHH 484079_A04 MGVSDVPRDLEVVAATPTSLLISWDAPA VTVRYYRITYGRQVQMYSDWGPLYIYAEFTVPGSKSTATISGLKPGVDYTITVYAV TGSGESPASSKPISINYRTEIDKPSQHH HHHH

Example 4: Biophysical Properties of PKE2 Adnectins

Size exclusion chromatography (SEC) was performed as described above ontwo of the PKE2 Adnectins, 2629_E06 and 2630_D02, identified in thescreen as being well behaved in Example 3. As shown in Table 2, bothPKE2 molecules were mostly monomeric.

Differential Scanning Calorimetry (DSC) analyses of the two PKE2Adnectins were performed to determine their respective Tm's as describedabove. As shown in Table 2, 2629_E06 and 2630_D02 had T_(M)s of 56 and57° C., respectively.

TABLE 2 PKE2 Binding SEC (% Adnectin loop monomer) T_(M) (° C.) 2270_C01CD 98% 64 2629_E06 CD >95% 56 2630_D02 CD >95% 57

Example 5: Characterization of the Binding of PKE2 Adnectins to SerumAlbumin of Various Species

The kinetics of binding to serum albumins by 2629_E06 and 2630_D02, aswell as that of a first generation north pole-based serum albuminbinding Adnectin, 1318_H04, were determined as described above. Inaddition, binding to albumin was carried out under various pH conditionsranging from pH 5.5 to pH 7.4. Neither 2629_E06 nor 2630_D02 showed pHdependent binding to human, rhesus, or mouse serum albumin, suggestingthey would maintain binding in the endosome. As shown in Table 3,1318_H04 had lower affinity for human, cynomolgus, and rhesus serumalbumin relative to 2629_E06 and 2630_D02, and further did not bind tomouse or rat serum albumin. Moreover, 1318_H04 exhibited a 10-foldweaker affinity for rhesus serum albumin relative to human serumalbumin, whereas the affinities of the PKE2 Adnectins for differentalbumin species were relatively equivalent.

Both PKE2 Adnectins, 2629_E06 and 2630_D02, showed substantially higheraffinity for all serum albumins tested relative to 1318_H04, asdiscussed above, with K_(D)S for human, cynomolgus, rhesus, and mouseserum albumin in the low nanomolar range. 2629_E06 also exhibited aK_(D) for rat serum albumin in the low nanomolar range, and 2630_D02exhibited a K_(D) for rat serum albumin of 200 nM.

Example 6: Competition of PKE2 Adnectins with hFcRn for Binding to HSA

Given that inhibiting the binding of HSA to the hFcRn receptor wouldprevent HSA recycling via hFcRn and reduce the long half-life of HSA,thus potentially reducing the magnitude of pharmacokinetic enhancement,the level of competition with hFcRn for binding to HSA was tested forthe PKE2 Adnectins using a competitive alpha screen, which is depictedin FIG. 1. Adnectins were serially diluted in assay buffer (50 mMAcetate/150 mM NaCl/0.1% Tween-20, pH 5.5+0.005% antifoam-204) to obtainthe desired final assay concentration range. A master mix of proteinsand alphascreen beads was prepared in assay buffer to obtain final assayconcentrations of 6.5 nM hFcRn-GST (BMS), 30 nM biotinylated human serumalbumin (Abcam) and 5 ug/ml each of Alphascreen Streptavidin donor beadsand AlphaLISA Glutathione acceptor beads (Perkin Elmer). 10 ul/well ofserially diluted Adnectin, followed by 10 ul/well of proteins+beadssolution were added to a 384-well small-volume assay plate (GreinerBio-one). The alphascreen beads and all transfers to the assay platewere protected from ambient light. The assay plate was sealed with anadhesive foil seal and incubated for 2-2.5 h with shaking at roomtemperature. The plate was read in a Synergy 4 reader (Biotek) withexcitation at 570 nm and emission at 680 nm. Average signal from controlwells without Adnectin was set as 0% inhibition and percent inhibitionof the FcRn-HSA interaction was calculated relative to that signal;average background signal from control wells without biotinylated HSAwas subtracted from all data points.

Table 4 and FIG. 2 show the results of the screen. Notably, 1318_H04more strongly competed with hFcRn for binding to HSA than the secondgeneration parent 2270_C01 Adnectin and PKE2 2629_E06 and 2630_D02Adnectins, suggesting that the PKE2 Adnectins may provide improved PKenhancement relative to 1318_H04.

Moreover, the domains on HSA bound by 1318_H04, 2629_E06, and 2630_D02were determined by SPR. As shown in Table 4, the 1318_H04 Adnectin boundto domain I of HSA, and 2270_C01, 2629_E06, and 2630_D02 bound to domainI-II of HSA but not domain I alone, suggesting that 1318_H04 and thePKE2 Adnectins bind to distinct epitopes on HSA. None of the Adnectinsin Table 4 bound to domain III of HSA, which domain is an importantinteraction site of HSA with FcRn.

Example 7: In Vivo Half-Life of Candidate PKE2 Adnectins

The PKE2 Adnectins 2629_E06 and 2630_D02 were prepared, purified andendotoxin removed. Wild-type mice (n=3/group) were injected with either2629_E06 or 2630_D02 at 1 mg/kg into the tail vein, and theconcentration in blood samples taken at intervals post-injection wasdetermined using a quantitative ELISA-based assay that was developed todetect the Adnectin in plasma samples. Specifically, Adnectin druglevels were measured in mouse plasma using the Mesoscale technologyplatform or standard colorimetric ELISAs. 2629_E06 and 2630_D02 werecaptured via an anti-His mAb (BMS) and detected using a rabbit anti-seradirected against the Adnectin scaffold in combination with a goatanti-rabbit HRP conjugated pAb. Alternatively, they were detected viaspecies-specific albumin bound to the Adnectin and a species specificanti-Albumin sulfo-tagged secondary pAb. The pharmacokinetic parametersof each Adnectin were determined using non-compartmental modeling withPhoenix WinNonlin software.

The pharmacokinetic profiles of 2629_E06 and 2630_D02 were compared asshown in FIG. 3 and Table 5. The half-life of 2629_E06 in mice plasmawas 33-41 hours, whereas the half-life of 2630_D02 was 35-39 hours.

TABLE 5 Cl_obs AUCall (mL/ Vz_obs (h * nmol/ MRTINF_pred Adnectin T1/2(h) h/kg) (mL/kg) L) (h) 2629_E06 36.5 ± 3.9 6.0 318 13150 45.8 2630_D0237.6 ± 2.4 4.5 243 17318 51.2

Example 8: Immunogenicity of PKE2 Adnectins

In silico prediction of HLA binding was evaluated using Epimatrixsoftware (Epivax). A comparison of the scores is shown in Table 6. ThePKE2 Adnectins 2629_E06 and 2630_D09 showed reduced in silico scoresrelative to 2270_C01. Additionally, in vitro proliferation of CD4+T-cells in response to 1318_H04, 2270_C01 and the PKE2 Adnectins wasevaluated as an ex vivo assessment of potential human immunogenicity.The Ficoll density gradient method was used to isolate peripheral bloodmononuclear cells (PBMC) from whole blood obtained from 40 independentdonors which were MHC Class II matched to the general population. Cellsfrom each donor were stored in liquid N2 following isolation and thawedprior to use. Cells from each donor were labeled with the fluorescentdye carboxyfluorescein succinimidyl ester (CFSE), and incubated with theAdnectins of interest for 7 days at 37° C. T cells were labeled with ananti CD4 antibody and proliferation was evaluated by flow cytometryusing the BD FACS Canto and FlowJo analysis software. Antigenicity of aprotein was calculated as the percentage of donors that showed asignificant increase in CD4+ proliferation.

Comparisons of the parent 2270_C01 and its two progeny 2629_E06 or2630_D02 revealed that the parent molecule had higher antigenicity (FIG.4 and Table 6), suggesting that the two progeny PKE2 Adnectins showreduced immunogenicity potential relative to the parent molecule.

TABLE 6 Strength Epi- Per- of matrix centage Response Re- Score Antigen-(Avg of Pos sponse CD CD Loop Adnectins icity responders) Index LoopSequence 1381_H04 0.08 5.23 0.39 2270_C01 0.73 6.82 4.95  1.71 WQVQMYSDWGPLYIYK 2629_E06 0.16 4.04 0.64 -5.63 REVQKYSD LGPLYIYQ 2630_D02 0.264.16 1.10 -5.98 REVQKYSD WGPLYIYN

Example 9: Effects of Single Cysteine Mutants of PKE2 Adnectins onBinding to Albumin

Single cysteine residues were incorporated at sites distinct from theHSA binding residues of the PKE-2 Adnectins in order to allow chemicalconjugation to therapeutic molecules of interest via standard maleimidechemistry. It was important to retain binding to serum albumin (and thusPK enhancement) in the context of a cysteine mutation, therefore theeffects of these mutations on binding to serum albumin of variousspecies were tested, using the 2629_E06 molecule as the basis formutation. The off-rate (k_(off)) of each of the mutants was analyzed viaan SPR-based assay, with albumins immobilized and Adnectins used asanalytes at 250 nM. As shown in Table 7, introduction of single cysteinemutations in 2629_E06 showed similar off-rates from serum albuminsacross various species as the parent 2629_E06 molecule, indicating thatbinding to serum albumin is retained in the context of these specificmutations. Therefore, any one of these cysteine mutants could serve as achemical conjugation partner for therapeutic molecules of interest andprovide PK enhancement.

Example 10: Biophysical Properties of Single Cysteine Mutants of2629_E06

The biophysical properties of the single cysteine mutants described inExample 9 were assessed, and are shown in Table 8. Every mutant yieldeda thermally stable and monomeric protein.

TABLE 8 Conc. Protein DSC (° C. @ Mutant Buffer (mg/ml) available (mg)SEC ASSA (M) 0.5 mg/ml) 2629_E06(A26C)-NYRTPH6 PBS 2.3 4.1 >99% 1.7265.5 monomer 2629_E06-NYRTPCH6 PBS 2.6 4.7 >99% 1.77 67.2 monomer2629_E06(T56C)-NYRTPH6 PBS 2.7 4.8 >99% 1.72 68.3 monomer2629_E06(T58C)-NYRTPH6 PBS 2.4 4.3 >99% 1.72 68.7 monomer2629_E06(A12C)-NYRTPH6 PBS 2.8 5.1 >99% 1.70 68.2 monomer2629_E06(S55C)-NYRTPH6 PBS 2.4 4.2 >99% 1.75 69.4 monomer2629_E06-NYRTPEDEDGCH6 PBS 0.7 1.3 >90% 1.73 70.5 monomerMGCSTSGVSD-2629_E06-NYRTPH6 PBS 1.6 2.8 >90% 1.71 67.8 monomer

Example 11: PKE2 Adnectin Tandem Molecule Modularity

One of the limitations of the north pole serum albumin binding Adnectinswas the lack of compatability for use in tandem with other ¹⁰Fn3proteins. Therefore, the compatability of the PKE2 Adnectins with other¹⁰Fn3 proteins was explored. The biophysical behavior of the PKE2Adnectins was tested when fused in tandem with an Adnectin specific fora different target. The PKE2 Adnectins were tested in both possibleconfigurations: in the N-terminal location (PKE2-X) and the C-terminallocation (X-PKE2). Size exclusion chromatography behavior was testedusing molecules obtained using the HTPP method. Fusions with the firstgeneration north pole-based 1318_H04 Adnectin was directly compared tofusions with the PKE2 Adnectins 2629_E06 and 2630_D02. Tested fusionpartners included a myostatin binding ¹⁰Fn3 domain (2987_H07; seeWO2014/043344), two PCSK9 binding ¹⁰Fn3 domains (2013_E01 and 2382_D09),and a EGFR binding ¹⁰Fn3 domain (1312_E01). The sequences of PCSK9Adnectins 2382_D09 and 2013_E01 can be found in WO2011/130354, hereinincorporated by reference. As shown in Table 9, both PKE2 Adnectinsmolecules consistently retained good biophysical behavior, as reflectedin the proportion of molecules with an SEC grading of A (i.e.,corresponding to ≧90% monomeric Adnectins), relative to the northpole-based 1318_H04 SABA molecule, in the context of a tandem Adnectin.PKE in the table refers to a serum albumin binding ¹⁰Fn3 domain (i.e.,PK-enhancing ¹⁰Fn3 domain). Ratios represent the # of clones withSEC=A/total # of clones tested. Tandems which were not generated aredenoted as “-”.

The data in FIG. 5 reproduce the data in Table 9 for the 2987_H07myostatin binding ¹⁰Fn3 domain and the 2013_E01 PCSK9 binding ¹⁰Fn3domain, with the exception that the various shades of gray reflect theability of the tandem molecules to still bind to HSA as determined inthe direct binding ELISA assay described above. The different shades ofgrey in FIG. 5 correspond to different EC₅₀ _(_)tandem Adnectin/EC₅₀_(_)monoAdnectin ratios for binding to HSA, with darker shadesrepresenting stronger binding of the tandem molecules to HSA. The datain FIG. 5 show that the PKE2-based tandem molecules had bettermonomericity (i.e., less prone to dimerization and aggregation) and HSAbinding (i.e., lost less HSA and RhSA binding) relative to the 1318_H04Adnectin. Similar patterns were observed with four additionaltarget-binding Adnectins. These data indicate that the PKE2 Adnectinsprovide a more stable and active binding partner for other ¹⁰Fn3proteins than the north pole serum albumin binding Adnectins.

Example 12: PCSK9-PKE2 Tandem Molecules Exhibit Good Potency in PCSK9Biochemical Assays, Low EpiMatrix Scores, Good Biophysical Propertiesand Cross-Species Albumin Binding

Various PCSK9-PKE2 tandem Adnectins were produced based on the PKE2Adnectin 2629_E06 and the PCSK9 Adnectin 2382_D09, as shown in Table 10.Each of the tandem molecules differ only by linker, and all were testedfor their biophysical and functional properties to ensure retention ofactivities of both the albumin-binding PKE2 and the PCSK9-bindingAdnectin. Cross-species albumin binding was determined using the ELISAmethod described above. The relative thermal stability was assessed byThermal Scanning Fluorescence (TSF). HTPP samples were normalized to 0.2mg/ml in PBS. 1 pd of Sypro orange dye diluted 1:40 with PBS was addedto 25 ul of each sample and the plate was sealed with a clear 96 wellmicroplate adhesive seal. Samples were scanned using a BioRad RT-PCRmachine by ramping the temperature from 25° C.-95° C., at a rate of 2degrees per minute. The data was analyzed using BioRad CFX manager 2.0software. The values obtained by TSF have been shown to correlate wellwith Tm values obtained by DSC over a melting range of 40° C. to 70° C.This is considered the acceptable working range for this technique. Aresult of ND (“No data”) is obtained when the slope of the transitioncurve is too small to allow its derivative peak (the rate of change influorescence with time) to be distinguished from noise.

The PCSK9:EGFA FRET assay measured the inhibition of PCSK9 binding tothe low density lipoprotein receptor (LDLR) epidermal growth factorprecursor homology domain (EGFA domain), using recombinant human PCSK9expressed in baculovirus and a synthetic 40-mer EGFA peptide(biotinylated). EGFA has been shown to represent the key interactingdomain of LDLR with PCSK9 (Kwon, H. J. et al., Proc. Natl. Acad. Sci.USA, 105(6): 1820-1825 (2008)). This assay used a PCSK9 C-terminaldomain binding mAb (mAb 4H5) labeled with Eu-chelate to provide FRETinteraction with biotinylated EGFA through the streptavidin/lallophycocyanin fluorophore complex. The PCSK9-LDLR FRET assay was runin a similar manner using the extracellular domain of LDLR in place ofthe EGFA peptide.

All tandem molecules had low immunogenicity (negative Epimatrix score),high monomericity (as assessed by SEC), acceptable relative thermalstability (TSF) and favorable cross-species albumin binding by ELISAassay. Moreover, the PCSK9-PKE2 tandem Adnectins retained good potencyin PCSK9 biochemical assays with IC₅₀s similar to the unformatted2382_D09 Adnectin.

TABLE 10 PCSK9: EGFA EGFA LDLR HuSA RhSA MuSA FRET 1 FRET 2 FRETPCSK9-PKE2 Conc EC50 EC50 EC50 (IC50, (IC50, (IC50, tandem LinkerEpimatrix (ug/mL) SEC TSF (nM) (nM) (nM) nM) nM) nM) 4472_E09 A −12.33490 A 58.5 23.8 19.8 7.0 5.81 4.81 1.3 4472_A11 B −21.2 4530 A 61 26.915.7 3.9 6.13 3.76 1.4 4472_H09 C −10.2 2410 A 62.5 27.8 26.8 4.0 9.667.17 3 4472_F04 D −18.8 3410 A 60.5 40.3 27.8 3.8 3.99 4.18 1.2 4472_C08E −14.4 2880 A 60 40.4 25.7 4.8 9.38 5.09 1.9 4472_F08 F −13.0 5050 A 6155.4 35.2 7.3 7.88 5.88 2.5 4472_F06 G NA 4450 A 61 58.9 40.8 11.3 4.503.42 1.1 4472_G10 H −15.7 1694 A 61 61.3 48.3 10.0 7.81 6.81 2.44472_E06 I −15.4 4810 A 60 67.3 40.1 4.2 7.67 5.68 1.6 4472_B10 J −5.41299 A 59.5 67.7 38.9 10.9 9.69 6.97 2.2 4472_B09 K −3.4 2230 A 59.569.2 45.1 9.8 9.36 6.95 2.4 4472_B11 L −11.5  835 A 59.0 70.1 54.0 13.56.54 5.03 2 4472_A06 M −7.0 3720 A 58 77.8 44.4 8.6 9.06 2.72 1.64472_D08 N −13.1 4140 A 60 80.2 54.1 9.5 6.32 5.22 1.7 4472_B05 O −6.2 639 A 57.5 85.1 59.5 9.3 6.66 5.55 1.9 4472_H11 P −1.7 1099 A nd 85.358.1 13.2 8.76 6.24 1.3 4472_E04 Q −5.6 1244 A 60.5 100.9 64.8 8.9 9.457.54 2 4472_E05 R −2.5   933*** A 54.5 102.2 63.1 13.1 5.66 4.35 1.54472_B03 S −7.9 1239 A 59 123.5 98.2 14.8 7.45 4.83 1.4 4472_D06 T −17.72850 A 61.5 139.2 93.1 15.6 8.12 6.51 1.8 4472_A04 U −6.6 1142 A nd143.7 84.0 16.7 5.14 4.44 1.1 4472_C06 PSTPPTPSPSTPPTPSPS −19.6 6760 A61 184.3 131.1 15.6 9.18 3.09 1.4 ADX_2382_D09 n/a 83 (DSC) n/a n/a n/a19.70 10.6 2.3 ADX_2629_E06 n/a 56 (DSC) 14.5 10.5 2.8 n/a n/a n/a

Example 13: Binding Kinetics of PCSK9-PKE2 Tandem Molecules to HumanPCSK9

PCSK9-PKE2 tandem Adnectin binding to immobilized human PCSK9 wasmeasured in the presence or absence of HSA by biolayer interferometry(Octet Red 96, using Superstreptavidin sensor tips, ForteBio, Menlo ParkCalif.). Association and dissociation events were captured in real timefor a series of Adnectin concentrations with biotinylated full-lengthPCSK9 captured on sensor tips. Binding curves were globally fit toproduce values for K_(D), k_(on), and k_(off). Tandem Adnectin-HSAcomplexes were pre-formed by incubating the tandem in excess and runningthe binding analysis in the presence of excess HSA. A complex betweenthe tandem Adnectin-HSA complexes with human PCSK9 was considered tohave formed when there was an increased apparent mass for the tandemAdnectins in the presence of HSA at the same concentration (see, e.g.,FIG. 6). As shown in Table 11, all tandem PCSK9-PKE2 molecules testedhad similar binding kinetics and potencies for PCSK9. A slight reductionwas seen for the association and somewhat faster dissociation for theHSA-Adnectin complex binding to huPCSK9.

TABLE 11 PCSK9: PKE2 Adnectin binding data table no HSA +HSA K_(D) (nM)k_(on) (1/Ms) k_(off) (1/s) K_(D) (nM) k_(on) (1/Ms) k_(off) (1/s)4472_F08 0.404 2.89E+05 1.18E−04 0.902 1.61E+05 1.48E−04 4472_E06 0.7242.81E+05 2.00E−04 1.003 2.07E+05 2.09E−04 4472_C06 0.515 3.36E+051.68E−04 1.547 1.97E+05 3.05E−04

Example 14: Characterization of the Binding of PCSK9-PKE2 TandemMolecules to Serum Albumin of Various Species

Affinities of PCSK9-PKE2 tandem molecules for serum albumin of variousspecies, alongside the affinity of the PKE2 Adnectin 2629_E06, wereassessed by Biacore analysis, as described in Example 2.

As shown in Table 12, all three PCSK9-PKE2 tandem molecules showedcomparable affinities to serum albumins across species, although theaffinities of the tandems were 5-7-fold weaker (with similar off-rates)compared to the 2629_E06 PKE2 Adnectin.

A similar experiment (under the conditions described in Example 2) wasperformed with the 4472_C06 tandem Adnectin without a 6× histidine tail(referred to as 5190_E01). As shown in Table 13, 5190_E01 bound to mouseserum albumin with a K_(D) similar to that of human, cynomolgus, andrhesus serum albumin, and bound to rat serum albumin with a K_(D) of 200nM.

The effects of pH on binding of PKE2 Adnectin 2629_E06 and PCSK9-PKE2tandem Adnectins 4472_C06, 4427_E06, and 4472_F08 were also tested. Asshown in Tables 14 and 15, all Adnectins tested showed pH insensitivebinding to serum albumin of various species.

Table 14.

Example 15: Dual Binding of Tandem PCSK9-PKE2 Adnectins to Albumins andPCSK9

The ability of the tandem PCSK9-PKE2 Adnectins to simultaneously bind toserum albumin and PCSK9 were assessed using SPR. It is likely that thetandem will be bound to albumin most of the time in vivo and thereforeit would be essential for activity of the PCSK9 Adnectin to be retainedwhen bound to albumin. Binding of the tandem PCSK9-PKE2 Adnectinssimultaneously to both targets was tested in the dual injection modewith a first injection of the tandem on to the immobilized albumin onthe chip surface, followed by a second injection of human PCSK9, andrecording the binding levels after a 3 min association phase for eachinjection. The increase in SPR binding signal upon injection of PCSK9 vsbuffer, indicates simultaneous binding of the tandem to HSA and PCSK9,as shown in FIG. 7. PCSK9 shows ˜40% of the expected binding level to500 nM or 1 uM tandem Adnectin pre-bound to HSA. As an additionalcontrol, PCSK9 shows no binding to PKE-2 alone (data not shown).

Example 16: In Vivo Clearance of PCSK9-PKE2 Adnectins in WT C57 BL/6Mice

The in vivo half-life of the tandem PCSK9-PKE2 Adnectin 4772_C06 wasdetermined in a 2 week single 2 mg/kg IV dose study in wild-type C57Bl/6 mice. Tandem Adnectin plasma levels were determined using theMesoScale Discovery platform. Biotinylated human PCSK9 was used tocapture the Adnectin and detection was via mouse serum albumin bound tothe tandem and an anti-mouse serum albumin sulfo-tagged secondary pAb.Non-compartmental analyses were performed using Phoenix WinNonlin 6.3(Pharsight Corporation, Mountain View, Calif.) using a plasma model andlinear up/log down calculation method. As shown in Table 16 and FIG. 8,the average half-life of the 4772_C06 tandem Adnectin was 16.7 hours.

TABLE 16 HL_Lambda_z Cl_obs Vss_obs AUCall AUCINF_obs AUC_%Extrap_obsMRTINF_obs strain dose mouse_ID (h) (mL/h/kg) (mL/kg) (h*nmol/L)(h*nmol/L) (%) (h) C57Bl/6 2 mg/kg N 3 3 3 3 3 3 3 Mean 16.7 4.39 92.018657 18787 0.69 21.0 SD 1.0 0.21 4.0 868 893 0.10 1.5 SE 0.6 0.12 2.3501 515 0.06 0.9 CV % 6 4.8 4.4 4.7 4.8 15.1 7.4

Example 17: PCSK9-PKE2 Tandem Adnectins Exhibit Robust PCSK9 TargetEngagement In Vivo

The pharmacodynamic activity of the PCSK9-PKE2 tandem Adnectin 4472_C06was assessed in a human PCSK9 transgenic mouse model that exhibitsnormal levels of human PCSK9. This model is a genomic hPCSK9 transgenic(BAC-transgenic) which is regulated in liver similarly to mouse PCSK9and which expresses near human-normal levels of hPCSK9 in plasma.Unbound hPCSK9 was evaluated following a single IP dose of PBS vehicleor 0.5 or 2 mg/kg tandem with 8 animals per group. An enzyme linkedimmunosorbance assays (ELISA) specific for free (unbound) human PCSK9that does not detect mouse PCSK9 was developed. The assay employedstreptavidin-pretreated 96-well plates coated with 2 μg/mL ofbiotinylated PCSK9-Adnectin 2013_E01 as capturing reagent. Plasmasamples frozen once only were diluted as appropriate in ELISA buffer (25mM Tris, 150 mM NaCl, pH 7.2 with 0.05% Tween-20 and 0.1% BSA), added towells and incubated for 1 hr at 20° C. Wells were then washed andincubated with 5 ug/mL of rabbit polyclonal anti-human PCSK9 IgG (BMScustom antibody produced by Lampire Biological Labs, Pipersville Pa.)for 1 hr, followed by processing for HRP-labeled anti-rabbit IgG withTMB by standard ELISA methods. Standard curves were generated usingpurified recombinant human PCSK9.

As shown in FIG. 9, analysis of the free hPCSK9 levels indicates potenttarget engagement by the PCSK9-PKE2 tandem Adnectin at both dosestested. Free hPCSK9 was inhibited in a dose dependent manner as shown bythe greater duration of response of the 2 mg/kg dose relative to the 0.5mg/kg dose. These data demonstrate in vivo activity of the PCSK9-PKE2tandem Adnectin.

Example 18: In Vivo Half-Life of PKE2 Mono-Adnectins and TandemPCSK9-PKE2 Adnectins in Cynomolgus Monkeys

Single dose PK/PD studies were conducted in normal lean female cynoswith comparisons of the PCSK9-PKE2 tandem between the PKE2 monoAdnectinor a PEGylated PCSK9 Adnectin comparator at molar dose equivalents, asindicated by the shading in Table 17 below. PKE2 Adnectin 2629_E06 orPCSK9-PKE2 tandem 5190_E01 Adnectin, or a PEGylated PCSK9 Adnectin(referred to as ATI-1476) as a comparator, were administered to cynos atthe indicated concentrations and routes (see Table 17 and FIG. 10), andblood plasma (K2EDTA) and serum samples were collected at time intervalsfor pharmacokinetic and pharmacodynamic assessment. Adnectin drug levelswere measured in cyno plasma using the Mesoscale technology platform.2629_E06 was captured via an anti-His mAb (BMS) and detected using cynoserum albumin bound to the Adnectin and an anti-cyno serum albuminsulfo-tagged secondary pAb. For tandem analyses, biotinylated humanPCSK9 was used to capture the Adnectin and detection was via cynoalbumin as described above. The PEGylated Adnectin ATI-1476 was capturedvia biotinylated hPCSK9 and detected via an anti-PEG mAb (Epitomics) inconjunction with a goat anti-rabbit sulfo-tagged pAb. Non-compartmentalanalyses were performed using Phoenix WinNonlin 6.3 (PharsightCorporation, Mountain View, Calif.) using a plasma model and linearup/log down calculation method.

As shown in Table 17, the plasma half-life of 2629_E06 and ATI-1476 wereequivalent at 112 hours. The half-life of 5190-E01 was shorter than thatof the PKE2 monoAdnectin and ranged from 60-82 hr following intravenousadministration. 5190_E01 exhibited dose proportional exposure between 3and 10 mg/kg intravenous doses (AUC_(ALL) ratio of 1.02). For allproteins tested, the volume of distribution was less than the plasmavolume, suggesting that the distribution of the PCSK9-PKE2 tandemAdnectin and PEGylated Adnectin was primarily limited to the vascularspace. Clearance was low in general and comparable across the variousdoses and formats. Subcutaneous bioavailability of the 5190_E01 tandemAdnectin was 41-49%.

TABLE 17 HL_Lambda_z Cl_obs Vss_obs AUCall AUCINF_obs AUC_%Extrap_obsMRTINF_obs Format Dose (h) (mL/h/kg) (mL/kg) (h*umol/L) (h*umol/L) (%)(h) PCSK9-PKE2  3 mg/kg SC — — —  92 ± 17  92 ± 17 0.54 ± 0.4 — tandem 3mg/kg IV  82 ± 5.1 0.55 ± 0.03 57 ± 4.3 222 ± 24 234 ± 12 5.45 ± 5.4104.6 ± 2.5 5190_E01 10 mg/kg IV   60 ± 6.5 0.58 ± 0.09 50 ± 6.8 754 ±98  769 ± 103 1.76 ± 1.4  87.3 ± 10.4 PCSK9-PEG 5 mg/kg IV 112 ± 7.60.46 ± 0.08 65 ± 10  1000 ± 14  1008 ± 14  0.70 ± 0.5 141.6 ± 7.6ATI-1476 PKE2 1.5 mg/kg IV   112 ± 7.3 0.37 ± 0.05 56 ± 5.2 305 ± 49 328± 45 6.92 ± 6.6  152 ± 8.2 2629_E06

The pharmacokinetics of parent 2270_C01 Adnectin was also tested incynomolgus monkeys in a separate study. Adnectin drug levels werequantified as described above for the mouse PK studies. As shown inTable 18 and in FIG. 11, the 2270_C01 Adnectin had a half-life of 83.5hours following a single IV bolus dose of 1 mg/kg.

TABLE 18 Vz_obs Cl_obs AUCINF_obs MRTINF_obs HL_Lambda_z (hr) (mL/kg)(mL/hr/kg) (hr * nmol/L) (hr) N 2 2 2 2 2 Mean 83.5 22.319 0.185419981.536 102.847 SD 8.632 2.819 0.004 9680.781 1.888 SE 6.104 1.9930.003 6845.346 1.335 Min 77.4 20.33 0.18 413136.19 101.51 Max 89.6 24.310.19 426826.88 104.18 CV % 10.3 12.6 2.3 2.3 1.8

Example 19: Tandem PCSK9-PKE2 Adnectin Functions as a PCSK9 Inhibitor inCynomolgus Monkeys

The cynomolgous monkey PK/PD study described above was evaluated for thepharmacodynamic effects of inhibiting PCSK9. Enzyme linkedimmunosorbance assays (ELISA) specific for cynomolgus PCSK9 weredeveloped. The free (unbound) PCSK9 assay employed the MesoScaleDiscovery platform and incorporated streptavidin-pretreated 96-well MSDplates coated with 2 μg/mL of biotinylated PCSK9-Adnectin 2013_E01 ascapturing reagent. Samples were diluted 1:4 with block and sulfo-taggedrabbit polyclonal anti-human PCSK9 IgG (BMS custom antibody produced byLampire Biological Labs, Pipersville Pa.), added to wells and incubatedfor 10 minutes at room temperature. Wells were then washed and readusing MSD 2× read buffer. The total PCSK9 ELISA assay was conductedsimilarly as described above except mAb-4H5 (BMS custom antibodyproduced by Lampire Biological Labs, Pipersville Pa.) was incorporatedas the capture antibody and the detection step was performed separatefrom the capture step. The mAb-4H5 binds the C-terminal domain of PCSK9,and when bound to the 96-well plates efficiently captures total PCSK9(both Adnectin-PCSK9 complex plus free PCSK9). The capture and detectionsteps for total PCSK9 were incubated for 1 hour. Standard curves weregenerated using purified recombinant human or cynomolgus PCSK9. Serumanalytes were assayed on a Siemens Advia 1800 Clinical Chemistry Systemusing standardized enzymatic procedures. LDL-cholesterol was assayed bythe direct LDL method (Roche Diagnostics). Other analytes tested were:aspartate aminotransferase; alanine aminotransferase; alkalinephosphatase; gamma glutamyltransferase; total bilirubin; blood ureanitrogen; creatinine; total cholesterol; trigylceride; high densitylipoprotein; low density lipoprotein; glucose; total protein; albumin;globulins; albumin/globulin ratio; calcium; inorganic phosphorus;sodium; potassium; chloride.

As shown in FIG. 12, 5190_E01 elicited the pharmacodynamic effects onunbound/free PCSK9, total PCSK9 and LDL-c that have been previouslyobserved with other PCSK9 Adnectin inhibitors. Specifically, rapidtarget engagement was observed in which free PCSK9 plummets tonon-detectable levels within 1 hour of dosing. LDL-c was lowered to ˜50%baseline as a result of PCSK9 inhibition, with maximum inhibition beingobserved in the 2-5 day time frame. Additionally, total PCSK9 rises asthe PCSK9-PKE2 Adnectin:PCSK9 complex accumulates. Upon complexdissociation and drug clearance, PCSK9 and LDL-c levels return tobaseline ˜15 days into the study. A similar trend is observed with thePEGylated PCSK9 Adnectin comparator. As shown in FIG. 13, 5190_E01exhibited similar robust LDL-c lowering at 10 mg/kg as the molar doseequivalent of the PEGylated PCSK9 Adnectin comparator.

Example 20: Dose Dependency in PCSK9 Target Engagement

A dose dependent response of free PCSK9 inhibition was observed in the 3and 10 mg/kg doses of the 5190_E01 as shown in FIG. 14. The 10 mg/kgdose exhibits a longer duration of PCSK9 target engagement than the 3mg/kg dose. This figure also illustrates equivalent PCSK9 targetengagement for the molar dose equivalents of the tandem and PEGylatedAdnectins. As expected, 2629_E06 does not modulate free PCSK9; anyobserved variation in free PCSK9 is likely due to diurnal rhythms andbaseline variability.

FIG. 15 illustrates the difference in effects of the tandem andPEGylated PCSK9 Adnectins on total PCSK9. Although the general trend isthe same, total PCSK9 peaks and returns to baseline more quickly in the5190_E01 dosed cynos relative to the PEGylated PCSK9 Adnectincomparator, suggesting different clearance mechanisms for thePCSK9:Adnectin drug complex depending on the PK enhancement methodemployed (renal clearance for the tandem vs. macrophage uptake for thePEGylated Adnectin). Again, 2629_E06 shows no pharmacodynamic effect inthis assay as expected.

Example 21: Tandem Format Exhibits Equivalent In Vitro ImmunogenicityResponse Relative to Components

In vitro assessment of potential immunogenicity was evaluated forseveral tandem PCSK9-PKE2 Adnectins using the T-cell proliferationassay, as described in Example 8.

As shown in FIG. 16, the percentage and magnitude of the immunogenicityresponse to tandem Adnectins is similar to the mono-Adnectin components(i.e., PCSK9 or PKE2; middle of FIG. 16). These results suggestminimal/no additional immunogenicity risk of tandem Adnectins vs.mono-Adnectins. Additionally, differences in the proliferative responseto the tandems are observed as a function of linker sequence joining thePCSK9 and PKE2 Adnectins. Relative to 4472_F08 and 4472_E06 tandemPCSK9-PKE2 Adnectins, the 4472_C06 tandem Adnectin showed the lowestimmunogenicity. One potential mechanism for these observed differencescould be differences in protein processing by the T-cells in response tothe linker sequences.

A summary of the properties of 4472_C06 is presented in Table 19 below.

Exemplary Embodiments

1. A polypeptide comprising a fibronectin type III tenth domain (¹⁰Fn3)wherein the ¹⁰Fn3 domain comprises a) AB, BC, CD, DE, EF, and FG loops,b) a CD loop with an altered amino acid sequence relative to thesequence of the corresponding CD loop of the human ¹⁰Fn3 domain, and c)wherein the polypeptide binds to human serum albumin with a K_(D) ofless than 500 nM.

2. The polypeptide of embodiment 1, wherein the ¹⁰Fn3 domain furtherbinds to one or more of rhesus serum albumin, cynomolgus serum albumin,mouse serum albumin, and rat serum albumin.

3. The polypeptide of embodiment 2, wherein the ¹⁰Fn3 domain binds torhesus serum albumin and cynomolgus serum albumin.

4. The polypeptide of embodiment 3, wherein the ¹⁰Fn3 domain bindsrhesus serum albumin and cynomolgus serum albumin with a K_(D) of lessthan 500 nM.

5. The polypeptide of embodiment 4, wherein the ¹⁰Fn3 domain bindsrhesus serum albumin and cynomolgus serum albumin with a K_(D) of lessthan 100 nM.

6. The polypeptide of embodiment 5, wherein the ¹⁰Fn3 domain bindsrhesus serum albumin and cynomolgus serum albumin with a K_(D) of lessthan 10 nM.

7. The polypeptide of any of the preceding embodiments, wherein the¹⁰Fn3 domain binds to mouse and rat serum albumin.

8. The polypeptide of embodiment 7, wherein the ¹⁰Fn3 domain bindsrhesus serum albumin and cynomolgus serum albumin with a K_(D) of lessthan 500 nM.

9. The polypeptide of embodiment 8, wherein the ¹⁰Fn3 domain bindsrhesus serum albumin and cynomolgus serum albumin with a K_(D) of lessthan 100 nM.

10. The polypeptide of embodiment 9, wherein the ¹⁰Fn3 domain bindsrhesus serum albumin and cynomolgus serum albumin with a K_(D) of lessthan 10 nM.

11. The polypeptide of any one of the preceding embodiments, wherein the¹⁰Fn3 domain binds to serum albumin at a pH range of 5.5 to 7.4.

12. The polypeptide of any one of the preceding embodiments, wherein the¹⁰Fn3 domain binds to domain I-II of HSA.

13. The polypeptide of any one of the preceding embodiments, wherein theserum half-life of the polypeptide in the presence of human serumalbumin is at least 30 hours.

14. The polypeptide of any one of the preceding embodiments, wherein theCD loop comprises an amino acid sequence according to the formulaG-X₁-X₂-V-X₃-X₄-X₅-S-X₆-X₇-G-X₈-X₉-Y-X₁₀-X₁₁-X₁₂-E, wherein,

-   -   (a) X₁ is selected from the group consisting of R or W;    -   (b) X₂ is selected from the group consisting of H, E, D, Y, or        Q;    -   (c) X₃ is selected from the group consisting of Q or H;    -   (d) X₄ is selected from the group consisting of I, K, M, Q, L,        or V;    -   (e) X₅ is selected from the group consisting of Y, F, or N;    -   (f) X₆ is selected from the group consisting of D, V, or E;    -   (g) X₇ is selected from the group consisting of L, W, or F;    -   (h) X₈ is selected from the group consisting of P or T;    -   (i) X₉ is selected from the group consisting of L or M;    -   (j) X₁₀ is selected from the group consisting of I or V;    -   (k) X₁₁ is selected from the group consisting of Y or F; and    -   (l) X₁₂ is selected from the group consisting of T, S, Q, N, or        A.        15. The polypeptide of embodiment 14, wherein:    -   (a) X₁ is R;    -   (b) X₂ is E;    -   (c) X₃ is Q;    -   (d) X₄ is K;    -   (e) X₅ is Y;    -   (f) X₆ is D;    -   (g) X₇ is L or W;    -   (h) X₈ is P;    -   (i) X₉ is L;    -   (j) X₁₀ is I;    -   (k) X₁₁ is Y; and    -   (l) X₁₂ is Q or N.

16. The polypeptide of embodiment 15, wherein X₁₀ is L and X₁₂ is Q.

17. The polypeptide of embodiment 15, wherein X₁₀ is W and X₁₂ is N.

18. The polypeptide of embodiment 14, wherein the CD loop comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:101-125.

19. The polypeptide of any one of the preceding embodiments, wherein theCD loop comprises the amino acid sequence set forth in SEQ ID NO: 106.

20. The polypeptide of any one of the preceding embodiments, wherein theCD loop comprises the amino acid sequence set forth in SEQ ID NO: 113.

21. A polypeptide of any one of the preceding embodiments, wherein thepolypeptide comprises an amino acid sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to the non-CD loop regions ofSEQ ID NOs: 23-100, 184-209 and 235-260.

22. The polypeptide of any one of the preceding embodiments, wherein thepolypeptide comprises an amino acid sequence that is at least 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs:23-100, 184-209 and 235-260.

23. A fusion polypeptide comprising a fibronectin type III tenth (¹⁰Fn3)domain and a heterologous protein, wherein the ¹⁰Fn3 domain comprises a)AB, BC, CD, DE, EF, and FG loops, b) a CD loop with an altered aminoacid sequence relative to the sequence of the corresponding loop of thehuman ¹⁰Fn3 domain, and c) wherein the polypeptide binds to human serumalbumin with a K_(D) of less than 500 nM.

24. The fusion polypeptide of embodiment 23, wherein the ¹⁰Fn3 domaincomprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 23-100,184-209 and 235-260.

25. The fusion polypeptide of embodiment 24, wherein the ¹⁰Fn3 domaincomprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 55, 81, 190 or 241.

26. The fusion polypeptide of embodiment 25, wherein the ¹⁰Fn3 domaincomprises an amino acid sequence of SEQ ID NO: 55, 81,190 or 241.

27. The fusion polypeptide of embodiment 24, wherein the ¹⁰Fn3 domaincomprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 62, 88, 197 or 248.

28. The fusion polypeptide of embodiment 27, wherein the ¹⁰Fn3 domaincomprises an amino acid sequence of SEQ ID NO: 62, 88, 197 or 248.

29. The fusion polypeptide of embodiment 23, wherein the heterologousprotein is a therapeutic moiety.

30. The fusion polypeptide of embodiment 23, wherein the heterologousprotein is a polypeptide comprising a ¹⁰Fn3 domain.

31. The fusion polypeptide of embodiment 30, wherein the ¹⁰Fn3 domainbinds to a target protein other than serum albumin.

32. The fusion polypeptide of embodiment 31, wherein the ¹⁰Fn3 domainbinds to PCSK9.

33. The fusion polypeptide of embodiment 32, wherein the ¹⁰Fn3 domaincomprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 167.

34. The fusion polypeptide of embodiment 33, wherein the ¹⁰Fn3 domaincomprises the amino acid sequence of SEQ ID NO: 167.

35. The fusion polypeptide of embodiment 23, wherein the fusionpolypeptide comprises an amino acid sequence at least 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 168,169 or 261.

36. The fusion polypeptide of embodiment 35, wherein the fusionpolypeptide comprises the amino acid sequence set forth in SEQ ID NO168, 169 or 261.

37. The fusion polypeptide of any one of embodiments 23-36, wherein theserum half-life of the polypeptide in the presence of mouse serumalbumin is at least 10 hours.

38. The fusion polypeptide of any one of embodiments 23-36, wherein theserum half-life of the polypeptide in the presence of cynomolgus serumalbumin is at least 50 hours.

39. A polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 23-125, 184-209 and 235-260, 168, and169.

40. A composition comprising a polypeptide of any one of the precedingembodiments and a carrier.

41. An isolated nucleic acid molecule encoding the polypeptide of anyone of embodiments 1-39.

42. The isolated nucleic acid molecule of embodiment 41, wherein thenucleic acid molecule has a sequence selected from the group consistingof SEQ ID NOs: 126-151 and 172 or a nucleotide sequence that is at least70%, 75%, 80%, 85%. 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

43. An expression vector comprising the nucleotide sequence ofembodiments 41 or 42.

44. A cell comprising a nucleic acid molecule of embodiment 41 or 42 oran expression vector of embodiment 43.

45. A method of producing the polypeptides of any one of embodiments1-39 comprising culturing the cell of embodiment 44 under conditionssuitable for expressing the polypeptide, and purifying the polypeptide.

TABLE 20 SUMMARY OF SEQUENCES SEQ ID Description Sequence 1 Wild-typeVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEF human ¹⁰Fn3TVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT domain 2 Wild-typeVSDVPRDLEVVAA(X)_(u)LLISW(X)_(v)YYRITY(X)_(w)FTV(X)_(x)ATISGLhuman ¹⁰Fn3 (X)_(y)YTITVYAV(X)_(z)ISINYRT domain w/ loop sequencesgenerically defined 3 N-terminal MGVSDVPRDL leader 4 N-terminalGVSDVPRDL leader 5 N-terminal X_(n)SDVPRDL leader 6 N-terminalX_(n)DVPRDL leader 7 N-terminal X_(n)VPRDL leader 8 N-terminal X_(n)PRDLleader 9 N-terminal X_(n)RDL leader 10 N-terminal X_(n)DL leader 11N-terminal MASTSG leader 12 C-terminal tail EIEK 13 C-terminal tailEGSGC 14 C-terminal tail EIEKPCQ 15 C-terminal tail EIEKPSQ 16C-terminal tail EIEKP 17 C-terminal tail EIEKPS 18 C-terminal tailEIEKPC 19 C-terminal tail EIDK 20 C-terminal tail EIDKPCQ 21C-terminal tail EIDKPSQ 22 6X His tail HHHHHH 23 PKE2 AdnectinMASTSGVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGWQVQMYSD 2270_C01 (aminoWGPLYIYKEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSK acid sequence)PISINYRTEGDKPSQHHHHHH 24 PKE2 AdnectinMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRHVQIYSDL 2629_A09 (aminoGPLYIYTEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP acid sequence)ISINYRTEIDKPSQHHHHHH 25 PKE2 AdnectinMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRHVHIYSDW 2629_A11 (aminoGPMYIYTEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP acid sequence)ISINYRTEIDKPSQHHHHHH 26 PKE2 AdnectinMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSVL 2629_C10 (aminoGPLYIYTEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP acid sequence)ISINYRTEIDKPSQHHHHHH 27 PKE2 AdnectinMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQMYSDL 2629_D09 (aminoGPLYVYSEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP acid sequence)ISINYRTEIDKPSQHHHHHH 28 PKE2 AdnectinMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKFSDW 2629_E05 (aminoGPLYIYTEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP acid sequence)ISINYRTEIDKPSQHHHHHH 29 PKE2 AdnectinMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDL 2629_E06 (aminoGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP acid sequence)ISINYRTEIDKPSQHHHHHH (also referred to as ATI-1490) 30 PKE2 AdnectinMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVHQYSDW 2629 F04 (aminoGPMYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP acid sequence)ISINYRTEIDKPSQHHHHHH 31 PKE2 AdnectinMGVSDVPRDLEVVAATPTSLLISWDAPAVTVXYYRITYGREVHKNSDW 2629_H01 (aminoGTLYIYTEFTVPGSKSTATISGLKPGVDYTITVXAVTGSGEXPASSKP acid sequence)ISINYRTEIDKXSQHHHHHH 32 PKE2 AdnectinMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDL 2629_H06 (aminoGPLYIYAEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP acid sequence)ISINYRTEIDKPSQHHHHHH 33 PKE2 AdnectinMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVHLYSDW 2629_H07 (aminoGPMYIYTEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP acid sequence)ISINYRTEIDKPSQHHHHHH 34 PKE2 AdnectinMGVSDVPRDLEVVATTPTSLLISWDAPAVTVRYYRITYGRHVQMYSDL 2630_A02 (aminoGPLYIFSEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP acid sequence)ISINYRTEIDKPSQHHHHHH 35 PKE2 AdnectinMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVHMYSDF 2630_A11 (aminoGPMYIYTEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP acid sequence)ISINYRTEIDKPSQHHHHHH 36 PKE2 AdnectinMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDW 2630_D02 (aminoGPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP acid sequence)ISINYRTEIDKPSQHHHHHH 37 PKE2 AdnectinMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQMYSDL 2630_D10 (aminoGPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP acid sequence)ISINYRTEIDKPSQHHHHHH 38 PKE2 AdnectinMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQMYSDL 2630_F04 (aminoGPLYIYTEFTVPGSKSTATISGLKPGVGYTITVYAVTGSGESPASSKP acid sequence)ISINYRTEIDKPSQHHHHHH 39 PKE2 AdnectinMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRHVQIYSDL 2630_G03 (aminoGPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP acid sequence)ISINYRTEIDKPSQHHHHHH 40 PKE2 AdnectinMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQIYSDW 2630_G10 (aminoGPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP acid sequence)ISINYRTEIDKPSQHHHHHH 41 PKE2 AdnectinMGVSDVPRDLEVVAATXTSLLISWDAPAVTVXYYRITYGREVQKYSDW 2630_H03 (aminoGPLYIYQEFTVPGSXSTATISGLKPGVDYTITVYAVTGSGESPASSKP acid sequence)ISINYRTEIDKXSQHHHHHH 42 PKE2 AdnectinMGVSDVPRDLEVVAATPTSLLISWDVPAVTVRYYRITYGRHVHLYSEF 2631_B04 (aminoGPMYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP acid sequence)ISINYRTEIDKPSQHHHHHH 43 PKE2 AdnectinMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRDVHMYSDW 2631_E03 (aminoGPMYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP acid sequence)ISINYRTEIDKPSQHHHHHH 44 PKE2 AdnectinMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRHVQIYSDW 2631_G01 (aminoGPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP acid sequence)ISINYRTEIDKPSQHHHHHH 45 PKE2 AdnectinMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRYVQLYSDW 2631_G03 (aminoGPMYIYTEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP acid sequence)ISINYRTEIDKPSQHHHHHH 46 PKE2 AdnectinMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRQVQVFSDL 2631_H09 (aminoGPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP acid sequence)ISINYRTEIDKPSQHHHHHH 47 PKE2 AdnectinMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRQVQIYSDW 2632_G01 (aminoGPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP acid sequence)ISINYRTEIDKPSQHHHHHH 48 PKE2 AdnectinMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRQVQMYSDW 4079_A04 (aminoGPLYIYAEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP acid sequence)ISINYRTEIDKPSQHHHHHH 49 2270_C01 w/o hisMASTSGVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGWQVQMYSD tag (amino acidWGPLYIYKEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSK sequence)PISINYRTEGDKPSQ 50 2629_A09 w/o hisMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRHVQIYSDL tag (amino acidGPLYIYTEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP sequence)ISINYRTEIDKPSQ 51 2629_A11 w/o hisMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRHVHIYSDW tag (amino acidGPMYIYTEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP sequence)ISINYRTEIDKPSQ 52 2629_C10 w/o hisMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSVL tag (amino acidGPLYIYTEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP sequence)ISINYRTEIDKPSQ 53 2629_D09 w/o his MGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQMYSDL tag (amino acidGPLYVYSEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP sequence)ISINYRTEIDKPSQ 54 2629_E05 w/o hisMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKFSDW tag (amino acidGPLYIYTEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP sequence)ISINYRTEIDKPSQ 55 2629_E06 w/o hisMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDL tag (amino acidGPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP sequence)ISINYRTEIDKPSQ 56 2629_F04 w/o hisMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVHQYSDW tag (amino acidGPMYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP sequence)ISINYRTEIDKPSQ 57 2629_H01 w/o hisMGVSDVPRDLEVVAATPTSLLISWDAPAVTVXYYRITYGREVHKNSDW tag (amino acidGTLYIYTEFTVPGSKSTATISGLKPGVDYTITVXAVTGSGEXPASSKP sequence)ISINYRTEIDKXSQ 58 2629_H06 w/o hisMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDL tag (amino acidGPLYIYAEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP sequence)ISINYRTEIDKPSQ 59 2629_H07 w/o hisMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVHLYSDW tag (amino acidGPMYIYTEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP sequence)ISINYRTEIDKPSQ 60 2630_A02 w/o hisMGVSDVPRDLEVVATTPTSLLISWDAPAVTVRYYRITYGRHVQMYSDL tag (amino acidGPLYIFSEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP sequence)ISINYRTEIDKPSQ 61 2630_A11 w/o hisMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVHMYSDF tag (amino acidGPMYIYTEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP sequence)ISINYRTEIDKPSQ 62 2630_D02 w/o hisMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDW tag (amino acidGPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP sequence)ISINYRTEIDKPSQ 63 2630_D10 w/o hisMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQMYSDL tag (amino acidGPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP sequence)ISINYRTEIDKPSQ 64 2630_F04 w/o hisMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQMYSDL tag (amino acidGPLYIYTEFTVPGSKSTATISGLKPGVGYTITVYAVTGSGESPASSKP sequence)ISINYRTEIDKPSQ 65 2630_G03 w/o hisMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRHVQIYSDL tag (amino acidGPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP sequence)ISINYRTEIDKPSQ 66 2630_G10 w/o hisMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQIYSDW tag (amino acidGPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP sequence)ISINYRTEIDKPSQ 67 2630_H03 w/o hisMGVSDVPRDLEVVAATXTSLLISWDAPAVTVXYYRITYGREVQKYSDW tag (amino acidGPLYIYQEFTVPGSXSTATISGLKPGVDYTITVYAVTGSGESPASSKP sequence)ISINYRTEIDKXSQ 68 2631_B04 w/o hisMGVSDVPRDLEVVAATPTSLLISWDVPAVTVRYYRITYGRHVHLYSEF tag (amino acidGPMYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP sequence)ISINYRTEIDKPSQ 69 2631_E03 w/o hisMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRDVHMYSDW tag (amino acidGPMYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP sequence)ISINYRTEIDKPSQ 70 2631_G01 w/o hisMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRHVQIYSDW tag (amino acidGPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP sequence)ISINYRTEIDKPSQ 71 2631_G03 w/o hisMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRYVQLYSDW tag (amino acidGPMYIYTEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP sequence)ISINYRTEIDKPSQ 72 2631_H09 w/o hisMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRQVQVFSDL tag (amino acidGPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP sequence)ISINYRTEIDKPSQ 73 2632_G01 w/o hisMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRQVQIYSDW tag (amino acidGPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP sequence)ISINYRTEIDKPSQ 74 4079_A04 w/o hisMGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRQVQMYSDW tag (amino acidGPLYIYAEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP sequence)ISINYRTEIDKPSQ 75 2270_C01 coreEVVAATPTSLLISWDAPAVTVRYYRITYGWQVQMYSDWGPLYIYKEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRT sequence) 76 2629_A09 coreEVVAATPTSLLISWDAPAVTVRYYRITYGRHVQIYSDLGPLYIYTEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRT sequence) 77 2629_A11 coreEVVAATPTSLLISWDAPAVTVRYYRITYGRHVHIYSDWGPMYIYTEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRT sequence) 78 2629_C10 coreEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSVLGPLYIYTEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRT sequence) 79 2629_D09 coreEVVAATPTSLLISWDAPAVTVRYYRITYGREVQMYSDLGPLYVYSEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRT sequence) 80 2629_E05 coreEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKFSDWGPLYIYTEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRT sequence) 81 2629_E06 coreEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRT sequence) 82 2629_F04 coreEVVAATPTSLLISWDAPAVTVRYYRITYGREVHQYSDWGPMYIYNEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRT sequence) 83 2629_H01 coreEVVAATPTSLLISWDAPAVTVXYYRITYGREVHKNSDWGTLYIYTEFT (amino acidVPGSKSTATISGLKPGVDYTITVXAVTGSGEXPASSKPISINYRT sequence) 84 2629_H06 coreEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYAEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRT sequence) 85 2629_H07 coreEVVAATPTSLLISWDAPAVTVRYYRITYGREVHLYSDWGPMYIYTEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRT sequence) 86 2630_A02 coreEVVATTPTSLLISWDAPAVTVRYYRITYGRHVQMYSDLGPLYIFSEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRT sequence) 87 2630_A11 coreEVVAATPTSLLISWDAPAVTVRYYRITYGREVHMYSDFGPMYIYTEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRT sequence) 88 2630_D02 coreEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDWGPLYIYNEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRT sequence) 89 2630_D10 coreEVVAATPTSLLISWDAPAVTVRYYRITYGREVQMYSDLGPLYIYNEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRT sequence) 90 2630_F04 coreEVVAATPTSLLISWDAPAVTVRYYRITYGREVQMYSDLGPLYIYTEFT (amino acidVPGSKSTATISGLKPGVGYTITVYAVTGSGESPASSKPISINYRT sequence) 91 2630_G03 coreEVVAATPTSLLISWDAPAVTVRYYRITYGRHVQIYSDLGPLYIYNEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRT sequence) 92 2630_G10 coreEVVAATPTSLLISWDAPAVTVRYYRITYGREVQIYSDWGPLYIYNEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRT sequence) 93 2630_H03 coreEVVAATXTSLLISWDAPAVTVXYYRITYGREVQKYSDWGPLYIYQEFT (amino acidVPGSXSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRT sequence) 94 2631_B04 coreEVVAATPTSLLISWDVPAVTVRYYRITYGRHVHLYSEFGPMYIYNEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRT sequence) 95 2631_E03 coreEVVAATPTSLLISWDAPAVTVRYYRITYGRDVHMYSDWGPMYIYQEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRT sequence) 96 2631_G01 coreEVVAATPTSLLISWDAPAVTVRYYRITYGRHVQIYSDWGPLYIYNEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRT sequence) 97 2631_G03 coreEVVAATPTSLLISWDAPAVTVRYYRITYGRYVQLYSDWGPMYIYTEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRT sequence) 98 2631_H09 coreEVVAATPTSLLISWDAPAVTVRYYRITYGRQVQVFSDLGPLYIYNEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRT sequence) 99 2632_G01 coreEVVAATPTSLLISWDAPAVTVRYYRITYGRQVQIYSDWGPLYIYNEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRT sequence) 1004079_A04 core EVVAATPTSLLISWDAPAVTVRYYRITYGRQVQMYSDWGPLYIYAEFT(amino acid VPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRT sequence) 1012629_A09 CD GRHVQIYSDLGPLYIYTE loop 102 2629_A11 CD GRHVHIYSDWGPMYIYTEloop 103 2629_C10 CD GREVQKYSVLGPLYIYTE loop 104 2629_D09 CDGREVQMYSDLGPLYVYSE loop 105 2629_E05 CD GREVQKFSDWGPLYIYTE loop 1062629_E06 CD GREVQKYSDLGPLYIYQE loop 107 2629_F04 CD GREVHQYSDWGPMYIYNEloop 108 2629_H01 CD GREVHKNSDWGTLYIYTE loop 109 2629_H06 CDGREVQKYSDLGPLYIYAE loop 110 2629_H07 CD GREVHLYSDWGPMYIYTE loop 1112630_A02 CD GRHVQMYSDLGPLYIFSE loop 112 2630_A11 CD GREVHMYSDFGPMYIYTEloop 113 2630_D02 CD GREVQKYSDWGPLYIYNE loop 114 2630_D10 CDGREVQMYSDLGPLYIYNE loop 115 2630_F04 CD GREVQMYSDLGPLYIYTE loop 1162630_G03 CD GRHVQIYSDLGPLYIYNE loop 117 2630_G10 CD GREVQIYSDWGPLYIYNEloop 118 2630_H03 CD GREVQKYSDWGPLYIYQE loop 119 2631_B04 CDGRHVHLYSEFGPMYIYNE loop 120 2631_E03 CD GRDVHMYSDWGPMYIYQE loop 1212631_G01 CD GRHVQIYSDWGPLYIYNE loop 122 2631_G03 CD GRYVQLYSDWGPMYIYTEloop 123 2631_H09 CD GRQVQVFSDLGPLYIYNE loop 124 2632_G01 CDGRQVQIYSDWGPLYIYNE loop 125 4079_A04 CD GRQVQMYSDWGPLYIYAE loop 1262270_C01 ATGGCTAGCACTAGTGGCGTGCCGCGCGACTTGGAAGTGGTTGCCGCG (nucleic acidACCCCGACGTCTCTGCTTATTAGCTGGGATGCACCTGCCGTCACAGTG sequence)AGATATTATCGCATTACATATGGTTGGCAGGTTCAGATGTACTCTGACTGGGGTCCGCTGTACATCTACAAAGAGTTCACGGTACCTGGGAGCAAGTCCACAGCTACCATCAGCGGTCTCAAACCTGGAGTTGATTACACCATTACGGTATACGCAGTCACCGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTATCGGACCGAAGGCGACAAACCATCCCAGCAC CATCACCACCACCACTGA 1272629_A09 ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACC (nucleic acidCCCACCAGCCTGCTGATCAGCTGGGATGCACCTGCCGTCACAGTGCGA sequence)TATTACCGCATCACTTACGGACGGCATGTTCAGATCTATTCTGACTTAGGCCCGCTGTACATCTACACAGAGTTCACTGTGCCTGGGAGCAAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCACTGA 1282629_A11 ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACC (nucleic acidCCCACCAGCCTGCTGATCAGCTGGGATGCACCTGCCGTCACAGTGCGA sequence)TATTACCGCATCACTTACGGTAGACACGTTCATATCTACTCAGACTGGGGTCCGATGTACATCTACACAGAGTTCACTGTGCCTGGGAGCAAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCACTGA 1292629_C10 ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACC (nucleic acidCCCACCAGCCTGCTGATCAGCTGGGATGCACCTGCCGTCACAGTGCGA sequence)TATTACCGCATCACTTACGGGAGAGAGGTTCAGAAATACTCTGTCTTGGGTCCACTGTACATATACACGGAGTTCACTGTGCCTGGGAGCAAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCACTGA 1302629_D09 ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACC (nucleic acidCCCACCAGCCTGCTGATCAGCTGGGATGCACCTGCCGTCACAGTGCGA sequence)TATTACCGCATCACTTACGGGAGGGAGGTTCAGATGTACTCTGACTTGGGTCCATTGTACGTATACAGCGAGTTCACTGTGCCTGGGAGCAAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCACTGA 1312629_E05 (nucleic ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCacid sequence) CCCACCAGCCTGCTGATCAGCTGGGATGCACCTGCCGTCACAGTGCGATATTACCGCATCACTTACGGTCGGGAGGTACAGAAGTTCTCGGACTGGGGTCCGCTGTACATCTACACAGAGTTCACTGTGCCTGGGAGCAAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCACTGA 1322629_E06 (nucleic ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCacid sequence) CCCACCAGCCTGCTGATCAGCTGGGATGCACCTGCCGTCACAGTGCGATATTACCGCATCACTTACGGCAGGGAGGTTCAGAAGTACTCGGACTTGGGTCCGTTGTACATCTACCAAGAGTTCACTGTGCCTGGGAGCAAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCACTGA 1332629_F04 (nucleic ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCacid sequence) CCCACCAGCCTGCTGATCAGCTGGGATGCACCTGCCGTCACAGTGCGATATTACCGCATCACTTACGGTAGGGAGGTTCATCAATACTCTGACTGGGGTCCGATGTACATCTACAACGAGTTCACTGTGCCTGGGAGCAAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCACTGA 1342629_H01 ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACC (nucleic acidCCCACCAGCCTGCTGATCAGCTGGGATGCACCTGCCGTCACAGTGCRA sequence)TATTACCGCATCACTTACGGTCGGGAGGTTCATAAGAACTCAGACTGGGGTACGCTGTACATCTACACAGAGTTCACTGTGCCTGGGAGCAAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTRTGCTGTCACTGGCTCTGGAGAGARCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAAMCATCCCAGCACCAT CACCACCACCACTGA 1352629_H06 ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACC (nucleic acidCCCACCAGCCTGCTGATCAGCTGGGATGCACCTGCCGTCACAGTGCGA sequence)TATTACCGCATCACTTACGGACGGGAGGTTCAGAAGTATTCAGACTTGGGTCCACTGTACATCTACGCAGAGTTCACTGTGCCTGGGAGCAAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCACTGA 1362629_H07 ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACC (nucleic acidCCCACCAGCCTGCTGATCAGCTGGGATGCACCTGCCGTCACAGTGCGA sequence)TATTACCGCATCACTTACGGGCGGGAGGTCCACCTGTACTCCGACTGGGGGCCGATGTACATCTACACAGAGTTCACTGTGCCTGGGAGCAAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCACTGA 1372630_A02 ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTACCACC (nucleic acidCCCACCAGCCTGCTGATCAGCTGGGATGCACCTGCCGTCACAGTGCGA sequence)TATTACCGCATCACTTACGGTAGGCACGTTCAAATGTACTCTGACCTTGGTCCGTTGTACATCTTCAGTGAGTTCACTGTGCCTGGGAGCAAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCACTGA 1382630_A11 ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACC (nucleic acidCCCACCAGCCTGCTGATCAGCTGGGATGCACCTGCCGTCACAGTGCGA sequence)TATTACCGCATCACTTACGGACGGGAGGTTCATATGTACTCTGACTTCGGTCCGATGTACATATACACAGAGTTCACTGTGCCTGGGAGCAAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCACTGA 1392630_D02 ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACC (nucleic acidCCCACCAGCCTGCTGATCAGCTGGGATGCACCTGCCGTCACAGTGCGA sequence)TATTACCGCATCACTTACGGTAGAGAAGTTCAGAAATACTCTGACTGGGGCCCGCTCTACATCTACAATGAGTTCACTGTGCCTGGGAGCAAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCACTGA 1402630_D10 ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACC (nucleic acidCCCACCAGCCTGCTGATCAGCTGGGATGCACCTGCCGTCACAGTGCGA sequence)TATTACCGCATCACTTACGGTCGGGAGGTTCAGATGTACTCGGACTTGGGTCCGCTCTACATCTACAACGAGTTCACTGTGCCTGGGAGCAAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCACTGA 1412630_F04 (nucleic ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCacid sequence) CCCACCAGCCTGCTGATCAGCTGGGATGCACCTGCCGTCACAGTGCGATATTACCGCATCACTTACGGTAGAGAGGTCCAGATGTACTCAGACTTGGGGCCGCTGTACATCTATACAGAGTTCACTGTGCCTGGGAGCAAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGGTTATACCATCACTGTGTATGCTGTCACTGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCACTGA 1422630_G03 ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACC (nucleic acidCCCACCAGCCTGCTGATCAGCTGGGATGCACCTGCCGTCACAGTGCGA sequence)TATTACCGCATCACTTACGGACGGCATGTTCAGATCTACTCCGACTTGGGTCCTCTGTATATCTACAATGAGTTCACTGTGCCTGGGAGCAAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCACTGA 1432630_G10 ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACC (nucleic acidCCCACCAGCCTGCTGATCAGCTGGGATGCACCTGCCGTCACAGTGCGA sequence)TATTACCGCATCACTTACGGTCGGGAGGTTCAAATATACTCTGACTGGGGTCCGCTGTATATATACAACGAGTTCACTGTGCCTGGGAGCAAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCACTGA 1442630_H03 ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACC (nucleic acidSCCACCAGCCTGCTGATCAGCTGGGATGCACCTGCCGTCACAGTGCSA sequence)TATTACCGCATCACTTACGGACGTGAAGTRCAGAAATACTCTGACTGGGGCCCGCTGTACATCTACCAAGAGTTCACTGTGCCTGGGAGCRAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAAMCATCCCAGCACCAT CACCACCACCACTGA 1452631_B04 ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACC (nucleic acidCCCACCAGCCTGCTGATCAGCTGGGATGTACCTGCCGTTACAGTGCGA sequence)TATTACCGCATCACTTACGGCAGGCACGTACATTTGTACTCGGAGTTCGGTCCGATGTATATCTACAACGAGTTCACTGTGCCTGGGAGCAAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCACTGA 1462631_E03 (nucleic ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCacid sequence) CCCACCAGCCTGCTGATCAGCTGGGATGCACCTGCCGTCACAGTGCGATATTACCGCATCACTTACGGTAGGGATGTCCACATGTACTCTGACTGGGGTCCGATGTACATATACCAAGAGTTCACTGTGCCTGGGAGCAAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCACTGA 1472631_G01 ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACC (nucleic acidCCCACCAGCCTGCTGATCAGCTGGGATGCACCTGCCGTCACAGTGCGA sequence)TATTACCGCATCACTTACGGTAGGCATGTTCAGATATACTCGGACTGGGGTCCGCTGTACATCTACAATGAGTTCACTGTGCCTGGGAGCAAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCACTGA 1482631_G03 ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACC (nucleic acidCCCACCAGCCTACTGATCAGCTGGGATGCACCTGCCGTCACAGTGCGA sequence)TATTACCGCATCACTTACGGAAGGTATGTTCAGCTATACTCTGACTGGGGTCCGATGTACATCTACACGGAGTTCACTGTGCCTGGGAGCAAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCACTGA 1492631_H09 ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACC (nucleic acidCCCACCAGCCTGCTGATCAGCTGGGATGCACCTGCCGTCACAGTGCGA sequence)TATTACCGCATCACTTACGGACGGCAAGTGCAAGTGTTCTCAGACTTGGGTCCGCTGTACATATACAACGAGTTCACTGTGCCTGGGAGCAAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCACTGA 1502632_G01 ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACC (nucleic acidCCCACCAGCCTGCTGATCAGCTGGGATGCACCTGCCGTCACAGTGCGA sequence)TATTACCGCATCACTTACGGTAGACAGGTGCAGATCTACTCTGACTGGGGACCGCTGTACATCTACAATGAGTTCACTGTGCCTGGGAGCAAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCACTGA 1514079_A04 ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACC (nucleic acidCCCACCAGCCTGCTGATCAGCTGGGATGCACCTGCCGTCACAGTGCGA sequence)TATTACCGCATCACTTACGGTAGGCAGGTACAGATGTACTCTGACTGGGGTCCACTTTACATCTACGCCGAGTTCACTGTGCCTGGGAGCAAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCAT CACCACCACCACTGA 152Linker PSTPPTPSPSTPPTPSPS 153 Linker GSGSGSGSGSGSGS 154 LinkerGGSGSGSGSGSGS 155 Linker GGSGSGSGSGSGSGSG 156 Linker GSEGSEGSEGSEGSE 157Linker GGSEGGSE 158 Linker GSGSGSGS 159 LinkerGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 160 Linker GGGGSGGGGSGGGGSGGGGSGGGGS161 Linker GGGGSGGGGSGGGGSG 162 Linker GPGPGPG 163 Linker GPGPGPGPGPG164 Linker PAPAPA 165 Linker PAPAPAPAPAPA 166 Linker PAPAPAPAPAPAPAPAPA167 PCSK9 ¹⁰Fn3 MGVSDVPRDLEVVAATPTSLLISWDAPAEGYGYYRITYGETGGNSPVQ domainEFTVPVSKGTATISGLKPGVDYTITVYAVEFDFPGAGYYHRPISINYR T 168 PCSK9-PKE2MGVSDVPRDLEVVAATPTSLLISWDAPAEGYGYYRITYGETGGNSPVQ tandem AdnectinEFTVPVSKGTATISGLKPGVDYTITVYAVEFDFPGAGYYHRPISINYR w/o his tagTEPSTPPTPSPSTPPTPSPSGVSDVPRDLEVVAATPTSLLISWDAPAV 5190_E01 (ATI-TVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDY 1676)TITVYAVTGSGESPASSKPISINYRTP 169 PCSK9-PKE2MGVSDVPRDLEVVAATPTSLLISWDAPAEGYGYYRITYGETGGNSPVQ tandem AdnectinEFTVPVSKGTATISGLKPGVDYTITVYAVEFDFPGAGYYHRPISINYR w/ his tagTEPSTPPTPSPSTPPTPSPSGVSDVPRDLEVVAATPTSLLISWDAPAV 4472_C06 (ATI-TVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDY 1574)TITVYAVTGSGESPASSKPISINYRTEHHHHHH 170 CD loopG-X₁-X₂-V-X₃-X₄-X₅-S-X₆-X₇-G-X₈-X₉-Y-X₁₀-X₁₁-X₁₂-E consensus 1713852_F10 ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGGACGCTCCGGCTGTTGACGGTCGATATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGGTTCTAAATCTACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTCCGTACGAATTCCATTTCCCGTACACTCATTACTCTTCTAAACCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCAC CACTGA 172 PCSK9-PKE2ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACC tandem AdnectinCCCACCAGCCTGCTGATCAGCTGGGACGCTCCGGCTGAAGGGTACGGT nucleic acidTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCCCTGTCCAG sequenceGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTT 5190_E01 (ATI-AAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCGAC 1676)TTCCCCGGCGCCGGTTACTACCATCGTCCAATTTCCATTAATTACCGGACCGAACCGAGCACACCTCCGACCCCGAGTCCGTCAACACCACCGACACCGTCACCGAGCGGAGTTTCTGACGTCCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGATCAGCTGGGATGCACCTGCCGTCACAGTGCGATATTACCGCATCACTTACGGCAGGGAGGTTCAGAAGTACTCGGACTTGGGTCCGTTGTACATCTACCAAGAGTTCACTGTGCCTGGGAGCAAGTCCACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATGCTGTCACTGGCTCTGGAGAGAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGCACACCGTGA 173 Linker PSPEPPTPEP 174 LinkerPSPEPPTPEPPSPEPPTPEP 175 Linker PSPEPPTPEPPSPEPPTPEPPSPEPPTPEP 176Linker PSPEPPTPEPPSPEPPTPEPPSPEPPTPEPPSPEPPTPEP 177 Linker EEEEDE 178Linker EEEEDEEEEDE 179 Linker EEEEDEEEEDEEEEDEEEEDE 180 LinkerEEEEDEEEEDEEEEDEEEEDEEEEDEEEEDE 181 Linker RGGEEKKKEKEKEEQEERETKTP 182Exemplary use of NYRTPGPSPEPPTPEP linker 183 Exemplary use ofPSPEPPTPEPGVSDV linker 184 2270_C01 coreEVVAATPTSLLISWDAPAVTVRYYRITYGWQVQMYSDWGPLYIYKEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTP sequence) with C-terminal proline 185 2629_A09 coreEVVAATPTSLLISWDAPAVTVRYYRITYGRHVQIYSDLGPLYIYTEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTP sequence) with C-terminal proline 186 2629_A11 coreEVVAATPTSLLISWDAPAVTVRYYRITYGRHVHIYSDWGPMYIYTEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTP sequence) with C-terminal proline 187 2629_C10 coreEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSVLGPLYIYTEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTP sequence) with C-terminal proline 188 2629_D09 coreEVVAATPTSLLISWDAPAVTVRYYRITYGREVQMYSDLGPLYVYSEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTP sequence) with C-terminal proline 189 2629_E05 coreEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKFSDWGPLYIYTEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTP sequence) with C-terminal proline 190 2629_E06 coreEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYQEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTP sequence) with C-terminal proline 191 2629_F04 coreEVVAATPTSLLISWDAPAVTVRYYRITYGREVHQYSDWGPMYIYNEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTP sequence) with C-terminal proline 192 2629_H01 coreEVVAATPTSLLISWDAPAVTVXYYRITYGREVHKNSDWGTLYIYTEFT (amino acidVPGSKSTATISGLKPGVDYTITVXAVTGSGEXPASSKPISINYRTP sequence) with C-terminal proline 193 2629_H06 coreEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLGPLYIYAEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTP sequence) with C-terminal proline 194 2629_H07 coreEVVAATPTSLLISWDAPAVTVRYYRITYGREVHLYSDWGPMYIYTEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTP sequence) with C-terminal proline 195 2630_A02 coreEVVATTPTSLLISWDAPAVTVRYYRITYGRHVQMYSDLGPLYIFSEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTP sequence) with C-terminal proline 196 2630_A11 coreEVVAATPTSLLISWDAPAVTVRYYRITYGREVHMYSDFGPMYIYTEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTP sequence) with C-terminal proline 197 2630_D02 coreEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDWGPLYIYNEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTP sequence) with C-terminal proline 198 2630_D10 coreEVVAATPTSLLISWDAPAVTVRYYRITYGREVQMYSDLGPLYIYNEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTP sequence) with C-terminal proline 199 2630_F04 coreEVVAATPTSLLISWDAPAVTVRYYRITYGREVQMYSDLGPLYIYTEFT (amino acidVPGSKSTATISGLKPGVGYTITVYAVTGSGESPASSKPISINYRTP sequence) with C-terminal proline 200 2630_G03 coreEVVAATPTSLLISWDAPAVTVRYYRITYGRHVQIYSDLGPLYIYNEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTP sequence) with C-terminal proline 201 2630_G10 coreEVVAATPTSLLISWDAPAVTVRYYRITYGREVQIYSDWGPLYIYNEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTP sequence) with C-terminal proline 202 2630_H03 coreEVVAATXTSLLISWDAPAVTVXYYRITYGREVQKYSDWGPLYIYQEFT (amino acidVPGSXSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTP sequence) with C-terminal proline 203 2631_B04 coreEVVAATPTSLLISWDVPAVTVRYYRITYGRHVHLYSEFGPMYIYNEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTP sequence) with C-terminal proline 204 2631_E03 coreEVVAATPTSLLISWDAPAVTVRYYRITYGRDVHMYSDWGPMYIYQEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTP sequence) with C-terminal proline 205 2631_G01 coreEVVAATPTSLLISWDAPAVTVRYYRITYGRHVQIYSDWGPLYIYNEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTP sequence) with C-terminal proline 206 2631_G03 coreEVVAATPTSLLISWDAPAVTVRYYRITYGRYVQLYSDWGPMYIYTEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTP sequence) with C-terminal proline 207 2631_H09 coreEVVAATPTSLLISWDAPAVTVRYYRITYGRQVQVFSDLGPLYIYNEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTP sequence) with C-terminal proline 208 2632_G01 coreEVVAATPTSLLISWDAPAVTVRYYRITYGRQVQIYSDWGPLYIYNEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTP sequence) with C-terminal proline 209 4079_A04 coreEVVAATPTSLLISWDAPAVTVRYYRITYGRQVQMYSDWGPLYIYAEFT (amino acidVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTP sequence) with C-terminal proline 210 C-terminal tail EIEPKSS 211 C-terminal tail EIDKPC212 C-terminal tail EIDKP 213 C-terminal tail EIDKPS 214 C-terminal tailEIDKPSQLE 215 C-terminal tail EIEDEDEDEDED 216 C-terminal tail EGSGS 217C-terminal tail EIDKPCQLE 218 C-terminal tail EIDKPSQHHHHHH 219C-terminal tail GSGCHHHHHH 220 C-terminal tail EGSGCHHHHHH 221C-terminal tail PIDK 222 C-terminal tail PIEK 223 C-terminal tail PIDKP224 C-terminal tail PIEKP 225 C-terminal tail PIDKPS 226 C-terminal tailPIEKPS 227 C-terminal tail PIDKPC 228 C-terminal tail PIEKPC 229C-terminal tail PIDKPSQ 230 C-terminal tail PIEKPSQ 231 C-terminal tailPIDKPCQ 232 C-terminal tail PIEKPCQ 233 C-terminal tail PHHHHHH 234C-terminal tail PCHHHHHH 235 2270_C01 w/o hisASTSGVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGWQVQMYSDW tag and N-terminalGPLYIYKEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKP methionine and w/ISINYRTEGDKPSQP C-terminal proline 236 2629_A09 w/o hisGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRHVQIYSDLG tag and N-terminalPLYIYTEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPI methionine and w/ SINYRTEIDKPSQP C-terminal proline 237 2629_A11 w/o hisGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRHVHIYSDWG tag and N-terminalPMYIYTEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPI methionine and w/SINYRTEIDKPSQP C-terminal proline 238 2629_C10 w/o hisGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSVLG tag and N-terminalPLYIYTEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPI methionine and w/SINYRTEIDKPSQP C-terminal proline 239 2629_D09 w/o hisGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQMYSDLG tag and N-terminalPLYVYSEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPI methionine and w/SINYRTEIDKPSQP C-terminal proline 240 2629_E05 w/o hisGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKFSDWG tag and N-terminalPLYIYTEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPI methionine and w/SINYRTEIDKPSQP C-terminal proline 241 2629_E06 w/o hisGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLG tag and N-terminalPLYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPI methionine and w/SINYRTEIDKPSQP C-terminal proline 242 2629_F04 w/o hisGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVHQYSDWG tag and N-terminalPMYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPI methionine and w/SINYRTEIDKPSQP C-terminal proline 243 2629_H01 w/o hisGVSDVPRDLEVVAATPTSLLISWDAPAVTVXYYRITYGREVHKNSDWG tag and N-terminalTLYIYTEFTVPGSKSTATISGLKPGVDYTITVXAVTGSGEXPASSKPI methionine and w/SINYRTEIDKXSQP C-terminal proline 244 2629_H06 w/o hisGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDLG tag and N-terminalPLYIYAEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPI methionine and w/SINYRTEIDKPSQP C-terminal proline 245 2629_H07 w/o hisGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVHLYSDWG tag and N-terminalPMYIYTEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPI methionine and w/SINYRTEIDKPSQP C-terminal proline 246 2630_A02 w/o hisGVSDVPRDLEVVATTPTSLLISWDAPAVTVRYYRITYGRHVQMYSDLG tag and N-terminalPLYIFSEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPI methionine and w/SINYRTEIDKPSQP C-terminal proline 247 2630_A11 w/o hisGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVHMYSDFG tag and N-terminalPMYIYTEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPI methionine and w/SINYRTEIDKPSQP C-terminal proline 248 2630_D02 w/o hisGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQKYSDWG tag and N-terminalPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPI methionine and w/SINYRTEIDKPSQP C-terminal proline 249 2630_D10 w/o hisGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQMYSDLG tag and N-terminalPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPI methionine and w/SINYRTEIDKPSQP C-terminal proline 250 2630_F04 w/o hisGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQMYSDLG tag and N-terminalPLYIYTEFTVPGSKSTATISGLKPGVGYTITVYAVTGSGESPASSKPI methionine and w/SINYRTEIDKPSQP C-terminal proline 251 2630_G03 w/o hisGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRHVQIYSDLG tag and N-terminalPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPI methionine and w/SINYRTEIDKPSQP C-terminal proline 252 2630_G10 w/o hisGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGREVQIYSDWG tag and N-terminalPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPI methionine and w/SINYRTEIDKPSQP C-terminal proline 253 2630_H03 w/o hisGVSDVPRDLEVVAATXTSLLISWDAPAVTVXYYRITYGREVQKYSDWG tag and N-terminalPLYIYQEFTVPGSXSTATISGLKPGVDYTITVYAVTGSGESPASSKPI methionine and w/SINYRTEIDKXSQP C-terminal proline 254 2631_B04 w/o hisGVSDVPRDLEVVAATPTSLLISWDVPAVTVRYYRITYGRHVHLYSEFG tag and N-terminalPMYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPI methionine and w/SINYRTEIDKPSQP C-terminal proline 255 2631_E03 w/o hisGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRDVHMYSDWG tag and N-terminalPMYIYQEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPI methionine and w/SINYRTEIDKPSQP C-terminal proline 256 2631_G01 w/o hisGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRHVQIYSDWG tag and N-terminalPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPI methionine and w/SINYRTEIDKPSQP C-terminal proline 257 2631_G03 w/o hisGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRYVQLYSDWG tag and N-terminalPMYIYTEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPI methionine and w/SINYRTEIDKPSQP C-terminal proline 258 2631_H09 w/o hisGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRQVQVFSDLG tag and N-terminalPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPI methionine and w/SINYRTEIDKPSQP C-terminal proline 259 2632_G01 w/o hisGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRQVQIYSDWG tag and N-terminalPLYIYNEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPI methionine and w/SINYRTEIDKPSQP C-terminal proline 260 4079_A04 w/o hisGVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGRQVQMYSDWG tag and N-terminalPLYIYAEFTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPI methionine and w/SINYRTEIDKPSQP C-terminal proline 261 PCSK9-PKE2GVSDVPRDLEVVAATPTSLLISWDAPAEGYGYYRITYGETGGNSPVQE tandem AdnectinFTVPVSKGTATISGLKPGVDYTITVYAVEFDFPGAGYYHRPISINYRT w/o his tag and N-EPSTPPTPSPSTPPTPSPSGVSDVPRDLEVVAATPTSLLISWDAPAVT terminalVRYYRITYGREVQKYSDLGPLYIYQEFTVPGSKSTATISGLKPGVDYT methionineITVYAVTGSGESPASSKPISINYRTP 5190_E01 (ATI- 1676) 262 Exemplary linker(PSPEPPTPEP)_(n) n = 1-10 263 Exemplary linker (EEEEDE)_(n)E n = 1-10

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A polypeptide comprising a fibronectin type III tenthdomain (¹⁰Fn3) wherein the ¹⁰Fn3 domain comprises a) AB, BC, CD, DE, EF,and FG loops, b) a CD loop with an altered amino acid sequence relativeto the sequence of the corresponding CD loop of the human ¹⁰Fn3 domain,c) wherein the polypeptide binds to human serum albumin with a K_(D) ofless than 500 nM, and d) wherein the ¹⁰Fn3 domain further binds to oneor more of rhesus serum albumin, cynomolgus serum albumin, mouse serumalbumin, and rat serum albumin with a K_(D) of less than 500 nM, forexample, less than 100 nM, or less than 10 nM.
 2. The polypeptide ofclaim 1, wherein the ¹⁰Fn3 domain binds to: a) rhesus serum albumin andcynomolgus serum albumin, b) mouse serum albumin and rat serum albumin,or c) rhesus serum albumin, cynomolgus serum albumin, mouse serumalbumin, and rat serum albumin.
 3. The polypeptide of any one of thepreceding claims, wherein the ¹⁰Fn3 domain binds to serum albumin at apH range of 5.5 to 7.4.
 4. The polypeptide of any one of the precedingclaims, wherein the ¹⁰Fn3 domain binds to domain I-II of HSA.
 5. Thepolypeptide of any one of the preceding claims, wherein the CD loopcomprises an amino acid sequence according to the formulaG-X₁-X₂-V-X₃-X₄-X₅-S-X₆-X₇-G-X₈-X₉-Y-X₁₀-X₁₁-X₁₂-E, wherein, (a) X₁ isselected from the group consisting of R or W; (b) X₂ is selected fromthe group consisting of H, E, D, Y, or Q; (c) X₃ is selected from thegroup consisting of Q or H; (d) X₄ is selected from the group consistingof I, K, M, Q, L, or V; (e) X₅ is selected from the group consisting ofY, F, or N; (f) X₆ is selected from the group consisting of D, V, or E;(g) X₇ is selected from the group consisting of L, W, or F; (h) X₈ isselected from the group consisting of P or T; (i) X₉ is selected fromthe group consisting of L or M; (j) X₁₀ is selected from the groupconsisting of I or V; (k) X₁₁ is selected from the group consisting of Yor F; and (l) X₁₂ is selected from the group consisting of T, S, Q, N,or A.
 6. The polypeptide of claim 5, wherein the CD loop comprises anamino acid sequence selected from the group consisting of SEQ ID NOs:106, 113, 101-105, 107-112, and 114-125.
 7. A polypeptide of any one ofthe preceding claims, wherein the polypeptide comprises an amino acidsequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to the non-CD loop regions of SEQ ID NOs: 81, 88, 23-80,82-87, 89-100, 184-209 and 235-260.
 8. The polypeptide of any one of thepreceding claims, wherein the polypeptide comprises an amino acidsequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to any one of SEQ ID NOs:: 81, 88, 23-80, 82-87, 89-100,184-209 and 235-260.
 9. A fusion polypeptide comprising a fibronectintype III tenth (¹⁰Fn3) domain and a heterologous protein, wherein the¹⁰Fn3 domain comprises a) AB, BC, CD, DE, EF, and FG loops, b) a CD loopwith an altered amino acid sequence relative to the sequence of thecorresponding loop of the human ¹⁰Fn3 domain, c) wherein the polypeptidebinds to human serum albumin with a K_(D) of less than 500 nM, andwherein the ¹⁰Fn3 domain further binds to one or more of rhesus serumalbumin, cynomolgus serum albumin, mouse serum albumin, and rat serumalbumin with a K_(D) of less than 500 nM, for example, less than 100 nM,or less than 10 nM.
 10. The fusion polypeptide of claim 9, wherein theheterologous protein is a therapeutic moiety.
 11. The fusion polypeptideof claim 9, wherein the heterologous protein is a polypeptide comprisinga ¹⁰Fn3 domain.
 12. The fusion polypeptide of claim 11, wherein the¹⁰Fn3 domain binds to a target protein other than serum albumin, such asPCSK9.
 13. The fusion polypeptide of claim 9, wherein the ¹⁰Fn3 domaincomprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 81, 88, 23-80, 82-87,89-100, 184-209 and 235-260.
 14. A polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 81, 88,23-80, 82-87, 89-100, 168-169, 184-209 and 235-261.
 15. A compositioncomprising a polypeptide of any one of the preceding claims and acarrier.
 16. An isolated nucleic acid molecule encoding the polypeptideof any one of claims 1-14.
 17. The isolated nucleic acid molecule ofclaim 16, wherein the nucleic acid molecule has a sequence selected fromthe group consisting of SEQ ID NOs: 126-151 and 172 or a sequence thatis at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical thereto.
 18. An expression vector comprising the nucleotidesequence encoding the polypeptide of any one of claims 1-14.
 19. A cellcomprising a nucleic acid molecule of claim 16 or 17 or expressionvector of claim
 18. 20. A method of producing the polypeptides of anyone of claims 1-14 comprising culturing the cell of claim 19 underconditions suitable for expressing the polypeptide, and purifying thepolypeptide.